Fusion proteins and methods for stimulating plant growth, protecting plants, and immobilizing bacillus spores on plants

ABSTRACT

The present invention is generally directed to fusion proteins containing a targeting sequence that targets the fusion protein to the exosporium of a  Bacillus cereus  family member. The invention also relates to recombinant  Bacillus cereus  family members expressing such fusion proteins and formulations containing the recombinant  Bacillus cereus  family members expressing the fusion proteins. Methods for stimulating plant growth, for protecting plants from pathogens, and for enhancing stress resistance in a plant by applying the recombinant  Bacillus cereus  family members or the formulations to plants or a plant growth medium are also described. The invention also relates to methods for immobilizing spores of a recombinant  Bacillus cereus  family member expressing a fusion protein on plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 15/846,487, filed Dec. 19, 2017, which is acontinuation of U.S. Non-Provisional patent application Ser. No.14/775,892, filed Sep. 14, 2015, now issued as U.S. Pat. No. 9,850,289,which is a U.S. National Stage Application of PCT Application No.PCT/US2014/030824, filed Mar. 17, 2014, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/799,262, filed Mar. 15, 2013.Each of the above-cited applications is incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

The present invention generally relates to fusion proteins containing atargeting sequence, an exosporium protein, or an exosporium proteinfragment that targets the fusion protein to the exosporium of a Bacilluscereus family member. The invention also relates to recombinant Bacilluscereus family members expressing such fusion proteins and formulationscontaining the recombinant Bacillus cereus family members expressing thefusion proteins. The invention further relates to methods forstimulating plant growth, for protecting plants from pathogens, and forenhancing stress resistance in plants by applying the recombinantBacillus cereus family members or the formulations to plants or a plantgrowth medium. The invention also relates to methods for immobilizingspores of a recombinant Bacillus cereus family member expressing afusion protein on plants or on plant matter.

BACKGROUND OF THE INVENTION

Within the zone surrounding a plant's roots is a region called therhizosphere. In the rhizosphere, bacteria, fungi, and other organismscompete for nutrients and for binding to the root structures of theplant. Both detrimental and beneficial bacteria and fungi can occupy therhizosphere. The bacteria, fungi, and the root system of the plant canall be influenced by the actions of peptides, enzymes, and otherproteins in the rhizosphere. Augmentation of soil or treatment of plantswith certain of these peptides, enzymes, or other proteins would havebeneficial effects on the overall populations of beneficial soilbacteria and fungi, create a healthier overall soil environment forplant growth, improve plant growth, and provide for the protection ofplants against certain bacterial and fungal pathogens. However, previousattempts to introduce peptides, enzymes, and other proteins into soil toinduce such beneficial effects on plants have been hampered by the lowsurvival of enzymes, proteins, and peptides in soil. Additionally, theprevalence of proteases naturally present in the soil leads todegradation of the proteins in the soil. The environment around theroots of a plant (the rhizosphere) is a unique mixture of bacteria,fungi, nutrients, and roots that has different qualities than that ofnative soil. The symbiotic relationship between these organisms isunique, and could be altered for the better with inclusion of exogenousproteins. The high concentration of fungi and bacteria in therhizosphere causes even greater degradation of proteins due toabnormally high levels of proteases and other elements detrimental toproteins in the soil. In addition, enzymes and other proteins introducedinto soil can dissipate away from plant roots quickly.

Thus, there exists a need in the art for a method for effectivelydelivering peptides, enzymes, and other proteins to plants (e.g., toplant root systems) and for extending the period of time during whichsuch molecules remain active. Furthermore, there exists a need in theart for a method of selectively targeting such peptides, enzymes, andproteins to the rhizosphere and to plant leaves and plant roots inparticular.

SUMMARY OF THE INVENTION

The present invention is directed to fusion proteins comprising at leastone plant growth stimulating protein or peptide, at least one protein orpeptide that enhances stress resistance in a plant, or at least oneplant binding protein or peptide. The plant growth stimulating proteinor peptide comprises a peptide hormone, a non-hormone peptide, or anenzyme involved in the production or activation of a plant growthstimulating compound. The fusion protein also comprises a targetingsequence, an exosporium protein, or an exosporium protein fragment. Thetargeting sequence, exosporium protein, or exosporium protein fragmentcan be: (a) a targeting sequence comprising an amino acid sequencehaving at least about 43% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 54%;(b) a targeting sequence comprising amino acids 1-35 of SEQ ID NO: 1;(c) a targeting sequence comprising amino acids 20-35 of SEQ ID NO: 1;(d) a targeting sequence comprising SEQ ID NO: 1; (e) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO: 2; (f) a targeting sequence comprising amino acids 1-27of SEQ ID NO: 3; (g) a targeting sequence comprising amino acids 12-27of SEQ ID NO: 3; (h) a targeting sequence comprising SEQ ID NO: 3; (i)an exosporium protein comprising an amino acid sequence having at least85% identity with SEQ ID NO: 4; (j) a targeting sequence comprisingamino acids 1-38 of SEQ ID NO: 5; (k) a targeting sequence comprisingamino acids 23-38 of SEQ ID NO: 5; (1) a targeting sequence comprisingSEQ ID NO: 5; (m) an exosporium protein comprising an amino acidsequence having at least 85% identity with SEQ ID NO: 6; (n) a targetingsequence comprising amino acids 1-28 of SEQ ID NO: 7; (o) a targetingsequence comprising amino acids 13-28 of SEQ ID NO: 7; (p) a targetingsequence comprising SEQ ID NO: 7; (q) an exosporium protein comprisingan amino acid sequence having at least 85% identity with SEQ ID NO: 8;(r) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 9;(s) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 9;(t) a targeting sequence comprising SEQ ID NO: 9; (u) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO: 10; (v) a targeting sequence comprising amino acids 1-33of SEQ ID NO:11; (w) a targeting sequence comprising amino acids 18-33of SEQ ID NO: 11; (x) a targeting sequence comprising SEQ ID NO: 11; (y)an exosporium protein comprising an amino acid sequence having at least85% identity with SEQ ID NO: 12; (z) a targeting sequence comprisingamino acids 1-33 of SEQ ID NO: 13; (aa) a targeting sequence comprisingamino acids 18-33 of SEQ ID NO: 13; (ab) a targeting sequence comprisingSEQ ID NO:13; (ac) an exosporium protein comprising an amino acidsequence having at least 85% identity with SEQ ID NO:14; (ad) atargeting sequence comprising amino acids 1-43 of SEQ ID NO: 15; (ae) atargeting sequence comprising amino acids 28-43 of SEQ ID NO: 15; (af) atargeting sequence comprising SEQ ID NO:15; (ag) an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO:16; (ah) a targeting sequence comprising amino acids 1-27 of SEQID NO: 17; (ai) a targeting sequence comprising amino acids 12-27 of SEQID NO: 17; (aj) a targeting sequence comprising SEQ ID NO:17; (ak) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO:18; (al) a targeting sequence comprising aminoacids 1-33 of SEQ ID NO: 19; (am) a targeting sequence comprising aminoacids 18-33 of SEQ ID NO: 19; (an) a targeting sequence comprising SEQID NO:19; (ao) an exosporium protein comprising an amino acid sequencehaving at least 85% identity with SEQ ID NO:20; (ap) a targetingsequence comprising amino acids 1-33 of SEQ ID NO: 21; (aq) a targetingsequence comprising amino acids 18-33 of SEQ ID NO: 21; (ar) a targetingsequence comprising SEQ ID NO:21; (as) an exosporium protein comprisingan amino acid sequence having at least 85% identity with SEQ ID NO:22;(at) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 23;(au) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 23;(av) a targeting sequence comprising SEQ ID NO:23; (aw) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO:24; (ax) a targeting sequence comprising amino acids 1-24of SEQ ID NO: 25; (ay) a targeting sequence comprising amino acids 9-24of SEQ ID NO: 25; (az) a targeting sequence comprising SEQ ID NO:25;(ba) an exosporium protein comprising an amino acid sequence having atleast 85% identity with SEQ ID NO:26; (bb) a targeting sequencecomprising amino acids 1-30 of SEQ ID NO: 27; (bc) a targeting sequencecomprising amino acids 15-30 of SEQ ID NO: 27; (bd) a targeting sequencecomprising SEQ ID NO:27; (be) an exosporium protein comprising an aminoacid sequence having at least 85% identity with SEQ ID NO:28; (bf) atargeting sequence comprising amino acids 1-33 of SEQ ID NO: 29; (bg) atargeting sequence comprising amino acids 18-33 of SEQ ID NO: 29; (bh) atargeting sequence comprising SEQ ID NO:29; (bi) an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO:30; (bj) a targeting sequence comprising amino acids 1-24 of SEQID NO: 31; (bk) a targeting sequence comprising amino acids 9-24 of SEQID NO: 31; (bl) a targeting sequence comprising SEQ ID NO:31; (bm) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO:32; (bn) a targeting sequence comprising aminoacids 1-15 of SEQ ID NO: 33; (bo) a targeting sequence comprising SEQ IDNO:33; (bp) an exosporium protein comprising an amino acid sequencehaving at least 85% identity with SEQ ID NO:34; (bq) a targetingsequence comprising amino acids 1-16 of SEQ ID NO: 35; (br) a targetingsequence comprising SEQ ID NO:35; (bs) an exosporium protein comprisingan amino acid sequence having at least 85% identity with SEQ ID NO:36;(bt) a targeting sequence comprising amino acids 1-29 of SEQ ID NO:43;(bu) a targeting sequence comprising amino acids 14-29 of SEQ ID NO: 43;(by) a targeting sequence comprising SEQ ID NO: 43; (bw) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO: 44; (bx) a targeting sequence comprising amino acids1-35 of SEQ ID NO: 45; (by) a targeting sequence comprising amino acids20-35 of SEQ ID NO: 45; (bz) a targeting sequence comprising SEQ ID NO:45; (ca) an exosporium protein comprising an amino acid sequence havingat least 85% identity with SEQ ID NO: 46; (cb) a targeting sequencecomprising amino acids 1-43 of SEQ ID NO: 47; (cc) a targeting sequencecomprising amino acids 28-43 of SEQ ID NO: 47; (cd) a targeting sequencecomprising SEQ ID NO: 47; (ce) an exosporium protein comprising an aminoacid sequence having at least 85% identity with SEQ ID NO: 48; (cf) atargeting sequence comprising amino acids 1-32 of SEQ ID NO: 49; (cg) atargeting sequence comprising amino acids 17-32 of SEQ ID NO: 49; (ch) atargeting sequence comprising SEQ ID NO: 49; (ci) an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO: 50; (cj) a targeting sequence comprising amino acids 1-33 of SEQID NO: 51; (ck) a targeting sequence comprising amino acids 18-33 of SEQID NO: 51; (cl) a targeting sequence comprising SEQ ID NO: 51; (cm) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO: 52; (cn) a targeting sequence comprising aminoacids 1-33 of SEQ ID NO: 53; (co) a targeting sequence comprising aminoacids 18-33 of SEQ ID NO: 53; (cp) a targeting sequence comprising SEQID NO: 53; (cq) an exosporium protein comprising an amino acid sequencehaving at least 85% identity with SEQ ID NO: 54; (cr) a targetingsequence comprising amino acids 1-30 of SEQ ID NO: 55; (cs) a targetingsequence comprising amino acids 15-30 of SEQ ID NO: 55; (ct) a targetingsequence comprising SEQ ID NO: 55; (cu) an exosporium protein comprisingan amino acid sequence having at least 85% identity with SEQ ID NO: 56;(cv) a targeting sequence comprising amino acids 1-130 of SEQ ID NO: 57;(cw) a targeting sequence comprising amino acids 115-130 of SEQ ID NO:57; (cx) a targeting sequence comprising SEQ ID NO: 57; (cy) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO: 58; (cz) an exosporium protein fragmentcomprising an amino acid sequence having at least 85% identity with SEQID NO: 59; (da) a targeting sequence comprising SEQ ID NO: 60; (db) atargeting sequence comprising SEQ ID NO: 61; (dc) a targeting sequencecomprising SEQ ID NO: 62; (dd) a targeting sequence comprising SEQ IDNO: 63; (de) a targeting sequence comprising SEQ ID NO: 64; (df) atargeting sequence comprising SEQ ID NO: 65; (dg) a targeting sequencecomprising SEQ ID NO: 66; (dh) a targeting sequence comprising SEQ IDNO: 67; (di) a targeting sequence comprising SEQ ID NO: 68; (dj) atargeting sequence comprising SEQ ID NO: 69; (dk) a targeting sequencecomprising SEQ ID NO: 70; (dl) an exosporium protein comprising an aminoacid sequence having at least 85% identity with SEQ ID NO: 71; (dm) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO: 72; (dn) an exosporium protein comprising anamino acid sequence having at least 85% identity with SEQ ID NO: 73;(do) an exosporium protein comprising an amino acid sequence having atleast 85% identity with SEQ ID NO: 74; (dp) an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO: 75; (dq) an exosporium protein comprising an amino acid sequencehaving at least 85% identity with SEQ ID NO: 76; (dr) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO: 77; (ds) an exosporium protein comprising an amino acidsequence having at least 85% identity with SEQ ID NO: 78; (dt) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO: 79; (du) an exosporium protein comprising anamino acid sequence having at least 85% identity with SEQ ID NO: 80;(dv) an exosporium protein comprising an amino acid sequence having atleast 85% identity with SEQ ID NO: 81; (dw) an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO: 82; (dx) an exosporium protein comprising an amino acid sequencehaving at least 85% identity with SEQ ID NO: 83; (dy) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO: 84; (dz) a targeting sequence comprising amino acids22-31 of SEQ ID NO: 1; (ea) a targeting sequence comprising amino acids22-33 of SEQ ID NO: 1; (eb) a targeting sequence comprising amino acids20-31 of SEQ ID NO: 1; (ec) a targeting sequence comprising amino acids14-23 of SEQ ID NO: 3; (ed) a targeting sequence comprising amino acids14-25 of SEQ ID NO: 3; or (ef) a targeting sequence comprising aminoacids 12-23 of SEQ ID NO: 3.

The present invention is also directed to fusion proteins comprising atargeting sequence, an exosporium protein, or an exosporium proteinfragment and at least one protein or peptide that protects a plant froma pathogen. The targeting sequence, an exosporium protein, or anexosporium protein fragment can be: (a) a targeting sequence consistingof an amino acid sequence consisting of 16 amino acids and having atleast about 43% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 54%; (b) atargeting sequence consisting of amino acids 1-35 of SEQ ID NO: 1; (c) atargeting sequence consisting of amino acids 20-35 of SEQ ID NO: 1; (d)a targeting sequence consisting of SEQ ID NO: 1; (e) a targetingsequence comprising SEQ ID NO: 60; (f) a targeting sequence comprisingamino acids 1-27 of SEQ ID NO: 3; (g) a targeting sequence comprisingamino acids 12-27 of SEQ ID NO: 3; (h) a targeting sequence comprisingSEQ ID NO: 3; (i) an exosporium protein comprising an amino acidsequence having at least 85% identity with SEQ ID NO: 4; (j) a targetingsequence comprising amino acids 1-38 of SEQ ID NO: 5; (k) a targetingsequence comprising amino acids 23-38 of SEQ ID NO: 5; (1) a targetingsequence comprising SEQ ID NO: 5; (m) an exosporium protein comprisingan amino acid sequence having at least 85% identity with SEQ ID NO: 6;(n) a targeting sequence comprising amino acids 1-28 of SEQ ID NO: 7;(o) a targeting sequence comprising amino acids 13-28 of SEQ ID NO: 7;(p) a targeting sequence comprising SEQ ID NO: 7; (q) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO: 8; (r) a targeting sequence comprising amino acids 1-24of SEQ ID NO: 9; (s) a targeting sequence comprising amino acids 9-24 ofSEQ ID NO: 9; (t) a targeting sequence comprising SEQ ID NO: 9; (u) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO: 10; (v) a targeting sequence comprising aminoacids 1-33 of SEQ ID NO:11; (w) a targeting sequence comprising aminoacids 18-33 of SEQ ID NO: 11; (x) a targeting sequence comprising SEQ IDNO: 11; (y) an exosporium protein comprising an amino acid sequencehaving at least 85% identity with SEQ ID NO: 12; (z) a targetingsequence comprising amino acids 1-33 of SEQ ID NO: 13; (aa) a targetingsequence comprising amino acids 18-33 of SEQ ID NO: 13; (ab) a targetingsequence comprising SEQ ID NO:13; (ac) an exosporium protein comprisingan amino acid sequence having at least 85% identity with SEQ ID NO:14;(ad) a targeting sequence comprising amino acids 1-43 of SEQ ID NO: 15;(ae) a targeting sequence comprising amino acids 28-43 of SEQ ID NO: 15;(af) a targeting sequence comprising SEQ ID NO:15; (ag) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO:16; (ah) a targeting sequence comprising amino acids 1-27of SEQ ID NO: 17; (ai) a targeting sequence comprising amino acids 12-27of SEQ ID NO: 17; (aj) a targeting sequence comprising SEQ ID NO:17;(ak) an exosporium protein comprising an amino acid sequence having atleast 85% identity with SEQ ID NO:18; (al) a targeting sequencecomprising amino acids 1-33 of SEQ ID NO: 19; (am) a targeting sequencecomprising amino acids 18-33 of SEQ ID NO: 19; (an) a targeting sequencecomprising SEQ ID NO:19; (ao) an exosporium protein comprising an aminoacid sequence having at least 85% identity with SEQ ID NO:20; (ap) atargeting sequence comprising amino acids 1-33 of SEQ ID NO: 21; (aq) atargeting sequence comprising amino acids 18-33 of SEQ ID NO: 21; (ar) atargeting sequence comprising SEQ ID NO:21; (as) an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO:22; (at) a targeting sequence comprising amino acids 1-24 of SEQID NO: 23; (au) a targeting sequence comprising amino acids 9-24 of SEQID NO: 23; (av) a targeting sequence comprising SEQ ID NO:23; (aw) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO:24; (ax) a targeting sequence comprising aminoacids 1-24 of SEQ ID NO: 25; (ay) a targeting sequence comprising aminoacids 9-24 of SEQ ID NO: 25; (az) a targeting sequence comprising SEQ IDNO:25; (ba) an exosporium protein comprising an amino acid sequencehaving at least 85% identity with SEQ ID NO:26; (bb) a targetingsequence comprising amino acids 1-30 of SEQ ID NO: 27; (bc) a targetingsequence comprising amino acids 15-30 of SEQ ID NO: 27; (bd) a targetingsequence comprising SEQ ID NO:27; (be) an exosporium protein comprisingan amino acid sequence having at least 85% identity with SEQ ID NO:28;(bf) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 29;(bg) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 29;(bh) a targeting sequence comprising SEQ ID NO:29; (bi) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO:30; (bj) a targeting sequence comprising amino acids 1-24of SEQ ID NO: 31; (bk) a targeting sequence comprising amino acids 9-24of SEQ ID NO: 31; (bl) a targeting sequence comprising SEQ ID NO:31;(bm) an exosporium protein comprising an amino acid sequence having atleast 85% identity with SEQ ID NO:32; (bn) a targeting sequencecomprising amino acids 1-15 of SEQ ID NO: 33; (bo) a targeting sequencecomprising SEQ ID NO:33; (bp) an exosporium protein comprising an aminoacid sequence having at least 85% identity with SEQ ID NO:34; (bq) atargeting sequence comprising amino acids 1-16 of SEQ ID NO: 35; (br) atargeting sequence comprising SEQ ID NO:35; (bs) an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO:36; (bt) a targeting sequence comprising amino acids 1-29 of SEQID NO:43; (bu) a targeting sequence comprising amino acids 14-29 of SEQID NO: 43; (by) a targeting sequence comprising SEQ ID NO: 43; (bw) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO: 44; (bx) a targeting sequence comprising aminoacids 1-35 of SEQ ID NO: 45; (by) a targeting sequence comprising aminoacids 20-35 of SEQ ID NO: 45; (bz) a targeting sequence comprising SEQID NO: 45; (ca) an exosporium protein comprising an amino acid sequencehaving at least 85% identity with SEQ ID NO: 46; (cb) a targetingsequence comprising amino acids 1-43 of SEQ ID NO: 47; (cc) a targetingsequence comprising amino acids 28-43 of SEQ ID NO: 47; (cd) a targetingsequence comprising SEQ ID NO: 47; (ce) an exosporium protein comprisingan amino acid sequence having at least 85% identity with SEQ ID NO: 48;(cf) a targeting sequence comprising amino acids 1-32 of SEQ ID NO: 49;(cg) a targeting sequence comprising amino acids 17-32 of SEQ ID NO: 49;(ch) a targeting sequence comprising SEQ ID NO: 49; (ci) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO: 50; (cj) a targeting sequence comprising amino acids1-33 of SEQ ID NO: 51; (ck) a targeting sequence comprising amino acids18-33 of SEQ ID NO: 51; (cl) a targeting sequence comprising SEQ ID NO:51; (cm) an exosporium protein comprising an amino acid sequence havingat least 85% identity with SEQ ID NO: 52; (cn) a targeting sequencecomprising amino acids 1-33 of SEQ ID NO: 53; (co) a targeting sequencecomprising amino acids 18-33 of SEQ ID NO: 53; (cp) a targeting sequencecomprising SEQ ID NO: 53; (cq) an exosporium protein comprising an aminoacid sequence having at least 85% identity with SEQ ID NO: 54; (cr) atargeting sequence comprising amino acids 1-30 of SEQ ID NO: 55; (cs) atargeting sequence comprising amino acids 15-30 of SEQ ID NO: 55; (ct) atargeting sequence comprising SEQ ID NO: 55; (cu) an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO: 56; (cv) a targeting sequence comprising amino acids 1-130 of SEQID NO: 57; (cw) a targeting sequence comprising amino acids 115-130 ofSEQ ID NO: 57; (cx) a targeting sequence comprising SEQ ID NO: 57; (cy)an exosporium protein comprising an amino acid sequence having at least85% identity with SEQ ID NO: 58; (cz) an exosporium protein fragmentconsisting of an amino acid sequence having at least 85% identity withSEQ ID NO: 59; (da) a targeting sequence comprising SEQ ID NO: 61; (db)a targeting sequence comprising SEQ ID NO: 62; (dc) a targeting sequencecomprising SEQ ID NO: 63; (dd) a targeting sequence comprising SEQ IDNO: 64; (de) a targeting sequence comprising SEQ ID NO: 65; (df) atargeting sequence comprising SEQ ID NO: 66; (dg) a targeting sequencecomprising SEQ ID NO: 67; (dh) a targeting sequence comprising SEQ IDNO: 68; (di) a targeting sequence comprising SEQ ID NO: 69; (dj) atargeting sequence comprising SEQ ID NO: 70; (dk) an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO: 71; (dl) an exosporium protein comprising an amino acid sequencehaving at least 85% identity with SEQ ID NO: 72; (dm) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO: 73; (dn) an exosporium protein comprising an amino acidsequence having at least 85% identity with SEQ ID NO: 74; (do) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO: 75; (dp) an exosporium protein comprising anamino acid sequence having at least 85% identity with SEQ ID NO: 76;(dq) an exosporium protein comprising an amino acid sequence having atleast 85% identity with SEQ ID NO: 77; (dr) an exosporium proteincomprising an amino acid sequence having at least 85% identity with SEQID NO: 78; (ds) an exosporium protein comprising an amino acid sequencehaving at least 85% identity with SEQ ID NO: 79; (dt) an exosporiumprotein comprising an amino acid sequence having at least 85% identitywith SEQ ID NO: 80; (du) an exosporium protein comprising an amino acidsequence having at least 85% identity with SEQ ID NO: 81; (dv) anexosporium protein comprising an amino acid sequence having at least 85%identity with SEQ ID NO: 82; (dw) an exosporium protein comprising anamino acid sequence having at least 85% identity with SEQ ID NO: 83;(dx) an exosporium protein comprising an amino acid sequence having atleast 85% identity with SEQ ID NO: 84; (dy) a targeting sequenceconsisting of amino acids 22-31 of SEQ ID NO: 1; (dz) a targetingsequence consisting of amino acids 22-33 of SEQ ID NO: 1; (ea) atargeting sequence consisting of amino acids 20-31 of SEQ ID NO: 1; (eb)a targeting sequence comprising amino acids 14-23 of SEQ ID NO: 3; (ec)a targeting sequence comprising amino acids 14-25 of SEQ ID NO: 3; or(ed) a targeting sequence comprising amino acids 12-23 of SEQ ID NO: 3.

The present invention is further directed to fusion proteins comprisinga targeting sequence, an exosporium protein, or an exosporium proteinfragment and at least one protein or peptide that protects a plant froma pathogen. The protein or peptide that protects a plant from a pathogencan comprise a harpin, an α-elastin, a β-elastin, a systemin, aphenylalanine ammonia-lyase, an elicitin, a defensin, a cryptogein, aflagellin protein, a flagellin peptide, a bacteriocin, a lysozyme, alysozyme peptide, a siderophore, a non-ribosomal active peptide, aconalbumin, an albumin, a lactoferrin, a lactoferrin peptide, or TasA.Alternatively, the protein or peptide that protects a plant from apathogen has insecticidal activity, helminthicidal activity, suppressesinsect or worm predation, or a combination thereof. Alternatively, theprotein that protects a plant from a pathogen comprises an enzyme. Thetargeting sequence, exosporium protein, or exosporium protein fragmentcan be any of the targeting sequences, exosporium proteins, orexosporium protein fragments listed above in paragraph [0005].

The present invention is also directed to fusion proteins comprising atleast one protein or peptide of interest and an exosporium protein. Theexosporium protein can be an exosporium protein comprising an amino acidsequence having at least 85% identity with any one of SEQ ID NOs: 71,75, 80, 81, 82, 83, and 84.

The invention further relates to a recombinant Bacillus cereus familymember that expresses any of the fusion proteins.

The invention is also directed to formulations comprising any of therecombinant Bacillus cereus family members and an agriculturallyacceptable carrier.

The present invention also relates to a method for stimulating plantgrowth. The method comprises introducing into a plant growth medium anyof the recombinant Bacillus cereus family members expressing a fusionprotein comprising at least one plant growth stimulating protein orpeptide, or any of the formulations comprising a recombinant Bacilluscereus family members expressing a fusion protein comprising at leastone plant growth stimulating protein or peptide. Alternatively, themethod comprises applying to a plant, a plant seed, or an areasurrounding a plant or a plant seed any of the recombinant Bacilluscereus family members expressing a fusion protein comprising at leastone plant growth stimulating protein or peptide, or any of theformulations comprising a recombinant Bacillus cereus family membersexpressing a fusion protein comprising at least one plant growthstimulating protein or peptide. The plant growth stimulating protein orpeptide is physically attached to the exosporium of the recombinantBacillus family member.

The present invention is also directed to a method for stimulating plantgrowth. The method comprises introducing a recombinant Bacillus cereusfamily member expressing a fusion protein into a plant growth medium orapplying a recombinant Bacillus cereus family member expressing a fusionprotein to a plant, a plant seed, or an area surrounding a plant or aplant seed. The fusion protein comprises at least one plant growthstimulating protein or peptide and a targeting sequence, an exosporiumprotein, or an exosporium protein fragment. The targeting sequence, anexosporium protein, or an exosporium protein fragment can be any ofthose listed above in paragraph [0005]. The plant growth stimulatingprotein or peptide is physically attached to the exosporium of therecombinant Bacillus family member.

The invention additionally relates to a method for protecting a plantfrom a pathogen or enhancing stress resistance in a plant. The methodcomprises introducing into a plant growth medium any of the recombinantBacillus cereus family member expressing a fusion protein comprising atleast one protein or peptide that protects a plant from a pathogen or atleast one protein or peptide that enhances stress resistance in a plant,or any of the formulations comprising any of the recombinant Bacilluscereus family member expressing a fusion protein comprising at least oneprotein or peptide that protects a plant from a pathogen or at least oneprotein or peptide that enhances stress resistance in a plant.Alternatively, the method comprises applying to a plant, a plant seed,or an area surrounding a plant any of the recombinant Bacillus cereusfamily members expressing a fusion protein comprising at least oneprotein or peptide that protects a plant from a pathogen or at least oneprotein or peptide that enhances stress resistance in a plant, or any ofthe formulations comprising any of the recombinant Bacillus cereusfamily members expressing a fusion protein comprising at least oneprotein or peptide that protects a plant from a pathogen or at least oneprotein or peptide that enhances stress resistance in a plant. Theprotein or peptide that protects a plant from a pathogen or the proteinor peptide that enhances stress resistance in a plant is physicallyattached to the exosporium of the recombinant Bacillus cereus familymember.

The present invention is also directed to a method for immobilizing arecombinant Bacillus cereus family member spore on a plant. The methodcomprises introducing into a plant growth medium any of the recombinantBacillus cereus family members expressing at least one plant bindingprotein or peptide, or any of the formulations comprising any of therecombinant Bacillus cereus family members expressing at least one plantbinding protein or peptide. Alternatively, the method comprises applyingto a plant, a plant seed, or an area surrounding a plant or a plant seedany of the recombinant Bacillus cereus family members expressing atleast one plant binding protein or peptide, or any of the formulationscomprising any of the recombinant Bacillus cereus family membersexpressing at least one plant binding protein or peptide. The plantbinding protein or peptide is physically attached to the exosporium ofthe recombinant Bacillus cereus family member.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of the amino acid sequence of theamino-terminal portion of Bacillus anthracis Sterne strain BclA and withthe corresponding region from various exosporium proteins from Bacilluscereus family members.

FIG. 2 shows exemplary fluorescent microscopy results for the expressionof fusion proteins containing various exosporium proteins linked to anmCherry reporter on the exosporium.

DEFINITIONS

When the articles “a,” “an,” “one,” “the,” and “said” are used herein,the mean “at least one” or “one or more” unless otherwise indicated.

The terms “comprising,” “including,” and “having” are intended to beinclusive and mean that there may be additional elements other than thelisted elements.

The term “bioactive peptide” refers to any peptide that exerts abiological activity. “Bioactive peptides” can be generated, for example,via the cleavage of a protein, peptide, proprotein, or preproprotein bya protease or peptidase.

An “enzyme involved in the production or activation of a plant growthstimulating compound” includes any enzyme that catalyzes any step in abiological synthesis pathway for a compound that stimulates plant growthor alters plant structure, or any enzyme that catalyzes the conversionof an inactive or less active derivative of a compound that stimulatesplant growth or alters plant structure to an active or more active formof the compound. Such compounds include, for example, but are notlimited to, small molecule plant hormones such as auxins and cytokinins,bioactive peptides, and small plant growth stimulating moleculessynthesized by bacteria or fungi in the rhizosphere (e.g.,2,3-butanediol).

The term “fusion protein” as used herein refers to a protein having apolypeptide sequence that comprises sequences derived from two or moreseparate proteins. A fusion protein can be generated by joining togethera nucleic acid molecule that encodes all or part of a first polypeptidewith a nucleic acid molecule that encodes all or part of a secondpolypeptide to create a nucleic acid sequence which, when expressed,yields a single polypeptide having functional properties derived fromeach of the original proteins.

The term “immobilizing a recombinant Bacillus cereus family member sporeon a plant” refers to the binding of a Bacillus cereus family memberspore to plant, e.g., to a root of a plant or to an aerial portion of aplant such as a leaf, stem, flower, or fruit, such that the spore ismaintained at the plant's root structure or aerial portion instead ofdissipating into the plant growth medium or into the environmentsurrounding the aerial portions of the plant.

A “plant growth medium” includes any material that is capable ofsupporting the growth of a plant.

A “plant immune system enhancer protein or peptide” as used hereinincludes any protein or peptide that has a beneficial effect on theimmune system of a plant.

The term “plant growth stimulating protein or peptide” as used hereinincludes any protein or peptide that increases plant growth in a plantexposed to the protein or peptide.

A “protein or peptide that protects a plant from a pathogen” as usedherein includes any protein or peptide that makes a plant exposed to theprotein or peptide less susceptible to infection with a pathogen.

A “protein or peptide that enhances stress resistance in a plant” asused herein includes any protein or peptide that makes a plant exposedto the protein or peptide more resistant to stress.

The term “plant binding protein or peptide” refers to any peptide orprotein capable of specifically or non-specifically binding to any partof a plant (e.g., roots or aerial portions of a plant such as leavesfoliage, stems, flowers, or fruits) or to plant matter.

The term “targeting sequence” as used herein refers to a polypeptidesequence that, when present as part of a longer polypeptide or aprotein, results in the localization of the longer polypeptide or theprotein to a specific subcellular location. The targeting sequencesdescribed herein result in localization of proteins to the exosporium ofa Bacillus cereus family member.

DESCRIPTION OF THE INVENTION

The present invention relates to fusion proteins containing a targetingsequence, an exosporium protein, or an exosporium protein fragment thattargets the fusion protein to the exosporium of a Bacillus cereus familymember and: (a) at least one plant growth stimulating protein orpeptide; (b) at least one protein or peptide that protects a plant froma pathogen; (c) at least one protein or peptide that enhances stressresistance of a plant; or (d) at least one plant binding protein orpeptide. When expressed in Bacillus cereus family member bacteria, thesefusion proteins are targeted to the exosporium layer of the spore andare physically oriented such that the protein or peptide is displayed onthe outside of the spore.

This Bacillus exosporium display (BEND) system can be used to deliverpeptides, enzymes, and other proteins to plants (e.g., to plant foliage,fruits, flowers, stems, or roots) or to a plant growth medium such assoil. Peptides, enzymes, and proteins delivered to the soil or anotherplant growth medium in this manner persist and exhibit activity in thesoil for extended periods of time. Introduction of recombinant Bacilluscereus family member bacteria expressing the fusion proteins describedherein into soil or the rhizosphere of a plant leads to a beneficialenhancement of plant growth in many different soil conditions. The useof the BEMD to create these enzymes allows them to continue to exerttheir beneficial results to the plant and the rhizosphere over the firstmonths of a plants life.

Targeting Sequences, Exosporium Proteins, and Exosporium ProteinFragments

For ease of reference, the SEQ ID NOs. for the peptide and proteinsequences referred to herein are listed in Table 1 below.

TABLE 1 Peptide and Protein Sequences Protein, protein fragment, ortargeting sequence (SEQ ID. NO) Sequence AA 1-41 of BclAMSNNNYSNGLNPDESLSASAFDPNLVGPTLPPIPPFTLPTG (B. anthracis Sterne)(SEQ ID NO: 1)* Full length BclAMSNNNYSNGLNPDESLSASAFDPNLVGPTLPPIPPFTLPTGPTGPFTTG (SEQ ID NO: 2)*PTGPTGPTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGPTGPTGPTGFTPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGPTFTGPTGPTGPTGATGLTGPTGPTGPSGLGLPAGLYAFNSGGISLDLGINDPVPFNTVGSQFFTGTAISQLDADTFVISETGFYKITVIANTATASVLGGLTIQVNGVPVPGTGSSLISLGAPFTIVIQAITQITTTPSLVEVIVTGLGLSLALGTSASIIIEKVA AA 1-33 ofMSEKYIILHGTALEPNLIGPTLPPIPPFTFPNG BetA/BAS3290 (B. anthracis Sterne)(SEQ ID NO: 3) Full lengthMSEKYIILHGTALEPNLIGPTLPPIPPFTFPNGPTGITGPTGATGFTGIGIT BetA/BAS3290GPTGVTGPTGIGITGPTGATGLGILPVFGTITTDVGIGFSVIVNTNINFTL (SEQ ID NO: 4)PGPVSGTTLNPVDNSIIINTTGVYSVSFSIVFVIQAISSSILNLTINDSIQFAIESRIGGGPGVRATSARTDLLSLNQGDVLRVRIREATGDIIYSNASLV VSKVD Met + AA 2-43 ofMVKVVEGNGGKSKIKSPLNSNFKILSDLVGPTFPPVPTGMTGIT BAS4623(B. anthracis Sterne) (SEQ ID NO: 5) Full length BAS4623VVKVVEGNGGKSKIKSPLNSNFKILSDLVGPTFPPVPTGMTGITGSTG (SEQ ID NO: 6)ATGNTGPTGETGATGSAGITGSTGPTGNTGGTGSTGPTGNTGATGSTGVTGSTGVTGSTGVTGSTGVTGSTGPTGETGGTGSTGVTGSTGATGSTGVTGNTGPTGSTGATGNTGSIGETGGTGSMGPTGETGVTGSTGGTGSTGVTGNTGPTGSTGVTGSTGVTGSTGPTGSTGVTGSTGPTGSTGVTGSTGVTGNMGPTGSTGVTGNTGSTGTTGATGETGPMGSTGATGTTGPTGETGETGETGGTGSTGPTGNTGATGSTGVTGSTGVTGSTGVTGETGPTGSTGATGNTGPTGETGGTGSTGATGSTGVTGNTGPTGSTGVTGNTGATGETGPTGNTGATGNTGPTGETGVTGSTGPTGETGVTGSTGPTGNTGATGETGATGSTGVTGNTGSTGETGPTGSTGPTGSTGATGVTGNTGPTGSTGATGATGSTGPTGSTGTTGNTGVTGDTGPTGATGVSTTATYAFANNTSGSVISVLLGGTNIPLPNNQNIGPGITVSGGNTVFTVANAGNYYIAYTINLTAGLLVSSRITVNGSPLAGTINSPTVATGSFSATIIASLPAGAAVSLQLFGVVALATLSTATPGATLTIIRLS AA 1-34 of BclBMKQNDKLWLDKGIIGPENIGPTFPVLPPIHIPTG (B. anthracis Sterne) (SEQ ID NO: 7)Full length BclB MKQNDKLWLDKGIIGPENIGPTFPVLPPIHIPTGITGATGATGITGATGP(SEQ ID NO: 8) TGTTGATGATGITGVTGATGITGVTGATGITGVTGATGITGVTGPTGITGATGPTGITGATGPAGITGVTGPTGITGATGPTGTTGVTGPTGDTGLAGATGPTGATGLAGATGPTGDTGATGPTGATGLAGATGPTGATGLTGATGATGATGGGAIIPFASGTTPALLVNAVLANTGTLLGFGFSQPGIAPGVGGTLTILPGVVGDYAFVAPRDGIITSLAGFFSATAALAPLTPVQIQMQIFIAPAASNTFTPVAPPLLLTPALPAIAIGTTATGIQAYNVPVVAGDKILVYVSLTGASPIAAVAGFVSAGLNIV AA 1-30 of BAS1882MDEFLSSAALNPGSVGPTLPPMQPFQFRTG (B. anthracis Sterne) (SEQ ID NO: 9)Full length BAS1882 MDEFLSSAALNPGSVGPTLPPMQPFQFRTGPTGSTGAKGAIGNTEPYW(SEQ ID NO: 10) HTGPPGIVLLTYDFKSLIISFAFRILPIS AA 1-39 of gene 2280MFDKNEIQKINGILQANALNPNLIGPTLPPIPPFTLPTG (B. weihenstephensis KBAB4)(SEQ ID NO: 11) Full length KBAB4MFDKNEIQKINGILQANALNPNLIGPTLPPIPPFTLPTGPTGVTGPTGVT gene 2280GPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGV (SEQ ID NO: 12)TGPTGVTGPTGVTGPTGVTGPTGVTGPTGETGPTGGTEGCLCDCCVLPMQSVLQQLIGETVILGTIADTPNTPPLFFLFTITSVNDFLVTVTDGTTTFVVNISDVTGVGFLPPGPPITLLPPTDVGCECECRERPIRQLLDAFIGSTVSLLASNGSIAADFSVEQTGLGIVLGTLPINPTTTVRFAISTCKITAVNIT PITMAA 1-39 of gene 3572 MFDKNEMKKTNEVLQANALDPNIIGPTLPPIPPFTLPTG(B. weihenstephensis KBAB4) (SEQ ID NO: 13) Full Length KBAB4MFDKNEMKKTNEVLQANALDPNIIGPTLPPIPPFTLPTGPTGPTGPTGP gene 3572TGPTGPTGPTGPTGPTGPTGPTGPTGPTGLTGPTGPTGLTGPTGLTGPT (SEQ ID NO: 14)GPTGLTGQTGSTGPTGATEGCLCDCCVFPMQEVLRQLVGQTVILATIADAPNVAPRFFLFNITSVNDFLVTVTDPVSNTTFVVNISDVIGVGFSLTVPPLTLLPPADLGCECDCRERPIRELLDTLIGSTVNLLVSNGSIATGFNVEQTALGIVIGTLPIPINPPPPTLFRFAISTCKITAVDITPTPTAT AA 1-49 ofMSRKDKFNRSRMSRKDRFNSPKIKSEISISPDLVGPTFPPIPSFTLPTG Exosporium LeaderPeptide (B. cereus VD200) (SEQ ID NO: 15) Full LengthMSRKDKFNRSRMSRKDRFNSPKIKSEISISPDLVGPTFPPIPSFTLPTGIT Exosporium LeaderGPTFNINFRAEKNVAQSFTPPADIQVSYGNIIFNNGGGYSSVTNTFTAPI PeptideNGIYLFSASIGFNPTLGTTSTLRITIRKNLVSVASQTGTITTGGTPQLEIT (SEQ ID NO: 16)TIIDLLASQTIDIQFSAAESGTLTVGSSNFFSGALLP AA 1-33 ofMNEEYSILHGPALEPNLIGPTLPSIPPFTFPTG Exosporium Leader Peptide(B. cereus VD166) (SEQ ID NO: 17) Full LengthMNEEYSILHGPALEPNLIGPTLPSIPPFTFPTGPTGITGPTGATGFTGIGIT Exosporium LeaderGPTGVTGPTGIGITGPTGATGPTGIGITGPTG Peptide (SEQ ID NO: 18) AA 1-39 ofMKNRDNNRKQNSLSSNFRIPPELIGPTFPPVPTGFTGIG hypothetical protein IKG_04663(B. cereus VD200) (SEQ ID NO: 19) Full LengthMKNRDNNRKQNSLSSNFRIPPELIGPTFPPVPTGFTGIGITGPTGPQGPT hypothetical proteinGPQGPRGLQGPMGEMGPTGPQGVQGIQGSVGPIGATGPEGQQGPQGL IKG_04663, partialRGPQGETGATGPGGVQGLQGPIGPTGATGAQGIQGIQGLQGPIGATGP (SEQ ID NO: 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 1-39 of YVTN β- MSDKHQMKKISEVLQAHALDPNLIGPPLPPITPFTFPTGpropeller protein (B. weihenstephensis KBAB4) (SEQ ID NO: 21)Full length YVTN β- MSDKHQMKKISEVLQAHALDPNLIGPPLPPITPFTFPTGSTGPTGSTGSpropeller protein TGPTGSTGNTGPTGPTGPPVGTNLDTIYVTNDISNNVSAIDGNTNTVLTKBAB4 TIPVGTNPVGVGVNSSTNLIYVVNNGSDNISVINGSTNTVVATIPVGTQ (SEQ ID NO: 22)PFGVGVNPSTNLIYVANRTSNNVSVIKGGTNTVLTTIPVGTNPVGVGVNSSTNLIYVTNEIPNSVSVIKGGTNTVVATIPVGLFPFGVGVNSLTNLIYVVNNSPHNVSVIDGNTNTVLTTISVGTSPVGVGVNLSTNLIYVANEVPNNISVINGNTNTVLTTIPVGTTPFEVGVNSSTNLIYVSNLNSNNVSVINGSANTVIATVPVGSVPRGIGVKP AA 1-30 of MDEFLSFAALNPGSIGPTLPPVPPFQFPTGhypothetical protein bcerkbab4_2363 (B. weihenstephensis KBAB4)(SEQ ID NO: 23) Full lengthMDEFLSFAALNPGSIGPTLPPVPPFQFPTGPTGSTGSTGPTGSTGSTGPT hypothetical proteinGFNLPAGPASITLTSNETTACVSTQGNNTLFFSGQVLVNGSPTPGVVV bcerkbab4_2363SFSFSNPSLAFMVPLAVITNASGNFTAVFLAANGPGTVTVTASLLDSP KBAB4 GTMASVTITIVNCP(SEQ ID NO: 24) AA 1-30 of MDEFLSSTALNPCSIGPTLPPMQPFQFPTGhypothetical protein bcerkbab4_2131 (B. weihenstephensis KBAB4)(SEQ ID NO: 25) Full lengthMDEFLSSTALNPCSIGPTLPPMQPFQFPTGPTGSTGTTGPTGSIGPTGNT hypothetical proteinGLTGNTGPTGITGPTGDTG bcerkbab4_2131 (SEQ ID NO: 26)AA 1-36 of triple helix MKERDRQNSLNSNFRISPNLIGPTFPPVPTGFTGIGrepeat containing collagen (B. weihenstephensis KBAB4) (SEQ ID NO: 27)Full length triple helixMKERDRQNSLNSNFRISPNLIGPTFPPVPTGFTGIGITGPTGPQGPTGPQ repeat-containingGPRGFQGPMGEMGPTGPQGVQGIQGPAGQMGATGPEGQQGPQGLRG collagen KBAB4PQGETGATGPQGVQGLQGPIGPTGATGAQGIQGIQGLQGPIGATGPEG (SEQ ID NO: 28)PQGIQGVQGVPGATGSQGIQGAQGIQGPQGPSGNTGATGVTGQGISGPTGITGPTGITGPSGGPPGPTGATGATGPGGGPSGSTGATGATGNTGVTGSAGVTGNTGSTGSTGETGAQGLQGIQGVQGPIGPTGPEGPQGIQGIPGPTGVTGEQGIQGVQGIQGITGATGDQGPQGIQGAIGPQGITGATGDQGPQGIQGVPGPTGDTGSQGVQGIQGPMGDIGPTGPEGPEGLQGPQGIQGVPGPAGATGPEGPQGIQGIQGPIGVTGPEGPQGIQGIQGIQGITGATGAQGATGVQGVQGNIGATGPEGPQGVQGTQGDIGPTGPMGPQGVQGIQGIQGPTGAQGVQGPQGIQGIQGPTGVTGDTGTTGATGEGTTGATGVTGPSGVTGPSGGPAGPTGPTGPSGPTGLTGPSGGPPGPTGATGVTGGVGDTGATGSTGVTGATGVTGATGATGLQGPQGIQGVQGDIGPTGPQGVQGPQGIQGITGATGDQGPQGIQGPQGIQGPTGPQGIQGGQGPQGIQGATGATGAQGPQGIQGIQGVQGPTGPQGPTGIQGVQGEIGPTGPQGVQGLQGPQGPTGDTGPTGPQGPQGIQGPTGATGATGSQGIQGPTGATGATGSQGIQGPTGATGATGATGATGATGATGATGVTGVSTTATYSFANNTSGSAISVLLGGTNIPLPNNQNIGPGITVSGGNTVFTVTNAGNYYIAYTINITAALLVSSRITVNGSPLAGTINSPAVATGSFNATIISNLAAGSAISLQLFGLLAVATLSTTTPGATLTIIRLS AA 1-39 ofVFDKNEIQKINGILQANALNPNLIGPTLPPIPPFTLPTG hypothetical proteinbmyco0001_21660 (B. mycoides 2048) (SEQ ID NO: 29) Full lengthVFDKNEIQKINGILQANALNPNLIGPTLPPIPPFTLPTGPTGGTGPTGVT hypothetical proteinGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGV bmyco0001_21660TGPTGVTGPTGVTGPTGVTGPTGGTEGCLCDCCVLPMQSVLQQLIGE (SEQ ID NO: 30)TVILGTIADTPNTPPLFFLFTITSVNDFLVTVTDGTTTFVVNISDVTGVGFLPPGPPITLLPPTDVGCECECRERPIRQLLDAFIGSTVSLLASNGSIAADFSVEQTGLGIVLGTLPINPTTTVRFAISTCKITAVNITPITM AA 1-30 ofMDEFLYFAALNPGSIGPTLPPVQPFQFPTG hypothetical protein bmyc0001_22540(B. mycoides 2048) (SEQ ID NO: 31) Full lengthMDEFLYFAALNPGSIGPTLPPVQPFQFPTGPTGSTGATGSTGSTGSTGP hypothetical proteinTGSTGSTGSTGSTGPTGPTGPTGSTGPTGPTGFNLPAGPASITLTSNETT bmyc0001_22540ACVSTQGNNTLFFSGQVLVNGSPTPGVVVSFSFSNPSLAFMVPLAVIT (SEQ ID NO: 32)NASGNFTAVFLAANGPGTVTVTASLLDSPGTMASVTITIVNCP AA 1-21 ofMDSKNIGPTFPPLPSINFPTG hypothetical protein bmyc0001_21510(B. mycoides 2048) (SEQ ID NO: 33) Full lengthMDSKNIGPTFPPLPSINFPTGVTGETGATGETGATGATGETGATGETG hypothetical proteinETGATGATGATGATGETGATGATGATGAAGATGETGATGETGATGE bmyc0001_21510TGATGETGATGVTGETGATGETGAAGETGITGVTGPTGETGATGETG (SEQ ID NO: 34)ATGATGITGATGITGVAGATGETGAAGETGPTGATGAIGAIGATGATGITGVTGATGETGAAGATGITGVTGATGETGAAGATGITGATGITGVAGATGITGPTGIPGTIPTTNLLYFTFSDGEKLIYTNADGIAQYGTTQILSPSEVSYINLFINGILQPQPFYEVTAGQLTLLDDEPPSQGSSIILQFIIIN AA 1-22 of collagenMIGPENIGPTFPILPPIYIPTG triple helix repeat protein (B. thuringiensis35646) (SEQ ID NO: 35) Full length collagenMIGPENIGPTFPILPPIYIPTGETGPTGITGATGETGPTGITGPTGITGATG triple helix repeatETGSTGITGATGETGSTGITGPIGITGATGETGPIGITGATGETGPTGITG proteinSTGITGLTGVTGLTGETGPIGITGPTGITGPTGVTGATGPTGGIGPITTT (SEQ ID NO: 36)NLLYYTFADGEKLIYTDTDGIPQYGTTNILSPSEVSYINLFVNGILQPQPLYEVSTGKLTLLDTQPPSQGSSIILQFIIIN AA 1-35 ofMSNNNIPSPFFFNNFNPELIGPTFPPIPPLTLPTG hypothetical proteinWP_69652 (B. cereus) (SEQ ID NO: 43) Full lengthMSNNNIPSPFFFNNFNPELIGPTFPPIPPLTLPTGPTGSTGATGATGPTGA hypothetical proteinTGPTGATGPTGATGATGSTGATGPTGATGTFSSANASIVTPAPQTVNN WP_69652LAPIQFTAPVLISKNVTFNGIDTFTIQIPGNYFFIGAVMTSNNQAGPVAV (SEQ ID NO: 44)GVGFNGIPVPSLDGANYGTPTGQEVVCFGFSGQIPAGTTINLYNISDKTISIGGATAAGSSIVAARLSFFRIS AA 1-41 ofMFSEKKRKDLIPDNFLSAPALDPNLIGPTFPPIPSFTLPTG exosporium leader WP016117717(B. cereus) (SEQ ID NO: 45) Full length exosporiumMFSEKKRKDLIPDNFLSAPALDPNLIGPTFPPIPSFTLPTGSTGPTGPTG leader WP016117717DTGPTGPTATICIRTDPDNGCSVAEGSGTVASGFASHAEACNTQAIGD (SEQ ID NO: 46)CSHAEGQFATASGTASHAEGFQTTASGFASHTEGSGTTADANFSHTEGINTIVDVLHPGSHIMGKNGTTRSSFSWHLANGLAVGPSLNSAVIEGVTGNLYLDGVVISPNAADYAEMFETIDGNLIDVGYFVTLYGEKIRKANANDDYILGVVSATPAMIADASDLRWHNLFVRDEWGRTQYHEVVVPE KKMAMEE AA 1-49 ofMTRKDKFNRSRISRRDRFNSPKIKSEILISPDLVGPTFPPIPSFTLPTG exosporium peptideWP002105192 (B. cereus) (SEQ ID NO: 47) Full length exosporiumMTRKDKFNRSRISRRDRFNSPKIKSEILISPDLVGPTFPPIPSFTLPTGVT peptide WP002105192GPTGNTGPTGITGPTGDTGPTGDTGPTGITGP (SEQ ID NO: 48) AA 1-38 ofMSRKDRFNSPKIKSEISISPDLVGPTFPPIPSFTLPTG hypothetical protein WP87353(B. cereus) (SEQ ID NO: 49) Full lengthMSRKDRFNSPKIKSEISISPDLVGPTFPPIPSFTLPTGITGPTGNTGPTGD hypothetical proteinTGPTGPTFNINFRAEKNGAQSFTPPADIQVSYGNIIFNNGGGYSSVTNT WP87353FTAPINGIYLFSANIGFNPTLGTTSTLRITIRKNLVSVASQTIDIQFSAAE (SEQ ID NO: 50)SGTLTVGSSNFF AA 1-39 of MKERDNKGKQHSLNSNFRIPPELIGPTFPPVPTGFTGIGexosporium peptide 02112369 (B. cereus) (SEQ ID NO: 51)Full length exosporiumMKERDNKGKQHSLNSNFRIPPELIGPTFPPVPTGFTGIGITGPTGPQGPT peptide 02112369GPQGPRGFQGPMGEMGPTGPQGVQGIQGPAGQMGATGPEGQQGPEG (SEQ ID NO: 52)LRGPVGATGATGLQGVQGIQGPIGSTGATGAQGIQGIQGLQGPIGATGPEGPQGIQGVQGLPGATGPQGVQGVQGVIGPQGPSGSTGGTGATGQGVTGPTGITGSTGVTGPSGGPPGPTGPTGATGPGGGPSGSTGVTGSTGNTGATGSPGVTGATGPTGSTGATGIQGSQGIQGIQGIQGPLGPTGPEGPQGIQGIPGPTGITGEQGIQGVQGIQGITGATGDQGT AA 1-39 ofMRERDNKRQQHSLNPNFRISPELIGPTFPPVPTGFTGIG exosporium protein WP016099770(B. cereus) (SEQ ID NO: 53) Full length exosporiumMRERDNKRQQHSLNPNFRISPELIGPTFPPVPTGFTGIGITGPTGPQGPT protein WP016099770GPQGPRGFQGPMGEMGPTGPQGVQGIQGPVGPIGATGPEGQQGPQGL (SEQ ID NO: 54)RGPQGETGATGPGGVQGLQGPIGPTGATGAQGVQGIQGLQGPIGATGPEGPQGIQGVQGLPGATGSQGIQGVQGIQGPQGPSGNTGATGATGQGITGPTGITGPTGITGPSGGPPGPTGPTGATGPGGGPSGSTGATGATGNTGATGNTGITGATGSTGPTGSTGAQGLQGIQGIQGPIGPTGPEGPQGIQGIPGPTGVTGEQGIQGVQGIQGITGATGDQGPQGIQGVIGAQGVTGATGDQGPQGIQGVPGPSGATGPQGVQGIQGPMGDIGPTGPEGPEGLQGPQGIQGVPGPVGATGPEGPQGIQGIQGVQGATGPQGPQGIQGIQGVQGITGA TG AA 1-36 ofMKNRDNKGKQQSNFRIPPELIGPTFPPVPTGFTGIG hypothetical protein YP006612525(B. thuringiensis) (SEQ ID NO: 55) Full lengthMKNRDNKGKQQSNFRIPPELIGPTFPPVPTGFTGIGITGPTGPQGPTGP hypothetical proteinQGPRGFQGPMGEMGPTGPQGVQGIQGPVGPIGATGPEGQQGAQGLR YP006612525GPQGETGATGPQGVQGLQGPIGPTGATGAQGIQGIQGLQGPIGATGPE (SEQ ID NO: 56)GPQGIQGVQGLPGATGPQGIQGAQGIQGTQGPSGNTGATGATGQGLTGPTGITGPTGITGPSGGPPGPTGPTGATGPGGGPSGSTGATGATGDTGATGSTGVTGATGAQGPQGVQGIQGPTGATGATGATGPQGIQGPQGIQGPTGATGATGSQGPTGNTGPTGSQGIQGPTGPTGAGATGATGATGATGVSTTATYAFANNTSGSIISVLLGGTNIPLPNNQNIGPGITVSGGNTVFTVANAGNYYIAYTINLTAGLLVSSRITVNGSPLAGTINSPAVAAGSFSATIIANLPAGAAVSLQLFGVIALATLSTATPGATLTIIRLS AA 1-136 ofMKFSKKSTVDSSIVGKRVVSKVNILRFYDARSCQDKDVDGFVDVGEL hypothetical proteinFTIFRKLNMEGSVQFKAHNSIGKTYYITINEVYVFVTVLLQYSTLIGGS TIGR03720YVFDKNEIQKINGILQANALNPNLIGPTLPPIPPFTLPTG (B. mycoides) (SEQ ID NO: 57)**Full length MKFSKKSTVDSSIVGKRVVSKVNILRFYDARSCQDKDVDGFVDVGELhypothetical protein FTIFRKLNMEGSVQFKAHNSIGKTYYITINEVYVFVTVLLQYSTLIGGSTIGR03720 YVFDKNEIQKINGILQANALNPNLIGPTLPPIPPFTLPTGPTGGTGPTGV(SEQ ID NO: 58)** TGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGGTEGCLCDCCVLPMQSVLQQLIGETVILGTIADTPNTPPLFFLFTITSVNDFLVTVTDGTTTFVVNISDVTGVGFLPPGPPITLLPPTDVGCECECRERPIRQLLDAFIGSTVSLLASNGSIAADFSVEQTGLGIVLGTLPINPTTTVRFAISTCKITAVNITPITM AA 1-196 of BclAMSNNNYSNGLNPDESLSASAFDPNLVGPTLPPIPPFTLPTGPTGPFTTG (B. anthracis Sterne)PTGPTGPTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGP (SEQ ID NO: 59)*TGPTGPTGFTPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGPTFTGPTGPTGPTGATGLTGPTGPTGPSGLG Met + AA 20-35 ofMAFDPNLVGPTLPPIPP BclA (B. anthracis Sterne) (SEQ ID NO: 60)Met + AA 12-27 of MALEPNLIGPTLPPIPP BetA/BAS3290 (B. anthracis Sterne)(SEQ ID NO: 61) Met + AA 18-33 of MALNPNLIGPTLPPIPP gene 2280(B. weihenstephensis KBAB4) (SEQ ID NO: 62) Met + AA 18-33 ofMALDPNIIGPTLPPIPP gene 3572 (B. weihenstephensis KBAB4) (SEQ ID NO: 63)Met + AA 12-27 of MALEPNLIGPTLPSIPP Exosporium Leader Peptide(B. cereus VD166) (SEQ ID NO: 64) Met + AA 18-33 of MALDPNLIGPPLPPITPYVTN β-propeller protein (B. weihenstephensis KBAB4) (SEQ ID NO: 65)Met + AA 9-24 of MALNPGSIGPTLPPVPP hypothetical protein bcerkbab4_2363(B. weihenstephensis KBAB4) (SEQ ID NO: 66) Met + AA 9-24 ofMALNPCSIGPTLPPMQP hypothetical protein bcerkbab4_2131(B. weihenstephensis KBAB4) (SEQ ID NO: 67) Met + AA 9-24 ofMALNPGSIGPTLPPVQP hypothetical protein bmyc0001_22540 (B. mycoides 2048)(SEQ ID NO: 68) Met + AA 9-24 of MALNPGSVGPTLPPMQP BAS1882(B. anthracis Sterne) (SEQ ID NO: 69) Met + AA 20-35 ofMALDPNLIGPTFPPIPS exosporium leader WP016117717 (B. cereus)(SEQ ID NO: 70) Full length InhAMKRKTPFKVFSSLAITTMLGCTFALGTSVAYAETTSQSKGSISTTPIDN (B. mycoides)NLIQEERLAEALKERGTIDQSASKEETQKAVEQYIEKKKGDQPNKEIL (SEQ ID NO: 71)PDDPAKEASDFVKKVKEKKMEEKEKVKKSVENASSEQTPSQNKKQLNGKVPTSPAKQAPYNGAVRTDKVLVLLVEFSDYKHNNIEQSPGYMYANDFSREHYQKMLFGNEPFTLFDGSKVKTFKQYYEEQSGGSYTTDGYVTEWLTVPGKAADYGADGKTGHDNKGPKGARDLVKEALKAAAEKGLDLSQFDQFDRYDTNGDGNQNEPDGVIDHLMVIHAGVGQEAGGGKLGDDAIWSHRSKLAQDPVAIEGTKSKVSYWDGKVAAHDYTIEPEDGAVGVFAHEFGHDLGLPDEYDTNYTGAGSPVEAWSLMSGGSWTGRIAGTEPTSFSPQNKDFLQKNMDGNWAKIVEVDYDKIKRGVGFPTYIDQSVTKSNRPGLVRVNLPEKSVETIKTGFGKHAYYSTRGDDMHTTLETPLFDLTKAANAKFDYKANYELEAECDFIEVHAVTEDGTKTLIDKLGDKVVKGDQDTTEGKWIDKSYDLSQFKGKKVKLQFDYITDPALTYKGFAMDNVNVTVDGKVVFSDDAEGQAKMKLNGFVVSDGTEKKPHYYYLEWRNYAGSDEGLKVGRGPVYNTGLVVWYADDSFKDNWVGRHPGEGFLGVVDSHPEAVVGNLNGKPVYGNTGLQIADAAFSLDQTPAWNVNSFTRGQFNYPGLPGVATFDDSKVYSNTQIPDAGRKVPQLGLKFQVVGQAD DKSAGAIWIRRFull length BAS 1141 MSCNENKHHGSSHCVVDVVKFINELQDCSTTTCGSGCEIPFLGAHNT(ExsY) ASVANTRPFILYTKAGAPFEAFAPSANLTSCRSPIFRVESVDDDSCAVL(B. anthracis Sterne) RVLSVVLGDSSPVPPTDDPICTFLAVPNARLVSTSTCITVDLSCFCAIQC(SEQ ID NO: 72) LRDVTI Full length BAS1144MFSSDCEFTKIDCEAKPASTLPAFGFAFNASAPQFASLFTPLLLPSVSPN (BxpB/ExsFA)PNITVPVINDTVSVGDGIRILRAGIYQISYTLTISLDNSPVAPEAGRFFLS(B. anthracis Sterne)LGTPANIIPGSGTAVRSNVIGTGEVDVSSGVILINLNPGDLIRIVPVELIG (SEQ ID NO: 73)TVDIRAAALTVAQIS Full length BAS1145MSCNCNEDHHHHDCDFNCVSNVVRFIHELQECATTTCGSGCEVPFLG (CotY)AHNSASVANTRPFILYTKAGAPFEAFAPSANLTSCRSPIFRVESIDDDD (B. anthracis Sterne)CAVLRVLSVVLGDTSPVPPTDDPICTFLAVPNARLISTNTCLTVDLSCF (SEQ ID NO: 74)CAIQCLRDVTI Full length BAS1140MEVGGTSVKNKNKSSTVGKPLLYIAQVSLELAAPKTKRIILTNFENED (B. anthracis Sterne)RKEESNRNENVVSSAVEEVIEQEEQQQEQEQEQEEQVEEKTEEEEQV (SEQ ID NO: 75)QEQQEPVRTVPYNKSFKDMNNEEKIHFLLNRPHYIPKVRCRIKTATISYVGSIISYRNGIVAIMPPNSMRDIRLSIEEIKSIDMAGF Full length ExsFBMKERSENMRSSSRKLTNFNCRAQAPSTLPALGFAFNATSPQFATLFTP (B. anthracis H9401)LLLPSTGPNPNITVPVINDTISTGTGIRIQVAGIYQISYTLTISLDNVPVTP (SEQ ID NO: 76)EAARFFLTLNSSTNIIAGSGTAVRSNIIGTGEVDVSSGVILINLNPGDLIQIVPVEVIGTVDIRSAALTVAQIR Full length InhA1MSKKPFKVLSSIALTAVLGLSFGAGTQSAYAETPVNKTATSPVDDHLI (B. thuringiensisPEERLADALKKRGVIDSKASETETKKAVEKYVENKKGENPGKEAAN HD74)GDQLTKDASDFLKKVKDAKADTKEKLNQPATGTPAATGPVKGGLNG (SEQ ID NO: 77)KVPTSPAKQKDYNGEVRKDKVLVLLVEYADFKHNNIDKEPGYMYSNDFNKEHYEKMLFGNEPFTLDDGSKIETFKQYYEEQSGGSYTVDGTVTKWLTVPGKAADYGADAPGGGHDNKGPKGPRDLVKDALKAAVDSGIDLSEFDQFDQYDVNGDGNKNQPDGLIDHLMIIHAGVGQEAGGGKLGDDAIWSHRWTVGPKPFPIEGTQAKVPYWGGKMAAFDYTIEPEDGAVGVFAHEYGHDLGLPDEYDTQYSGQGEPIEAWSIMSGGSWAGKIAGTTPTSFSPQNKEFFQKTIGGNWANIVEVDYEKLNKGIGLATYLDQSVTKSARPGMIRVNLPDKDVKTIEPAFGKQYYYSTKGDDLHTKMETPLFDLTNATSAKFDFKSLYEIEAGYDFLEVHAVTEDGKQTLIERLGEKANSGNADSTNGKWIDKSYDLSQFKGKKVKLTFDYITDGGLALNGFALDNASLTVDGKVVFSDDAEGTPQLKLDGFVVSNGTEKKKHNYYVEWRNYAGADNALKFARGPVFNTGMVVWYADSAYTDNWVGVHPGHGFLGVVDSHPEAIVGTLNGKPTVKSSTRFQIADAAFSFDKTPAWKVVSPTRGTFTYDGLAGVPKFDDSKTYINQQIPDAGRILPKLGLKFEVVGQADDNSAG AVRLYR Full length ExsJMKHNDCFDHNNCNPIVFSADCCKNPQSVPITREQLSQLITLLNSLVSAI (B. cereus ATCCSAFFANPSNANRLVLLDLFNQFLIFLNSLLPSPEVNFLKQLTQSIIVLLQ 10876)SPAPNLGQLSTLLQQFYSALAQFFFALDLIPISCNSNVDSATLQLLFNL (SEQ ID NO: 78)LIQLINATPGATGPTGPTGPTGPTGPAGTGAGPTGATGATGATGPTGATGPAGTGGATGATGATGVTGATGATGATGPTGPTGATGPTGATGATGATGPTGATGPTGATGLTGATGAAGGGAIIPFASGTTPSALVNALVANTGTLLGFGFSQPGVALTGGTSITLALGVGDYAFVAPRAGTITSLAGFFSATAALAPISPVQVQIQILTAPAASNTFTVQGAPLLLTPAFAAIAIGSTASGIIAEAIPVAAGDKILLYVSLTAASPIAAVAGFVSAGINIV Full length ExsHMKHNDCFGHNNCNNPIVFTPDCCNNPQTVPITSEQLGRLITLLNSLIAA (B. cereus)IAAFFANPSDANRLALLNLFTQLLNLLNELAPSPEGNFLKQLIQSIINLL (SEQ ID NO: 79)QSPNPNLGQLLSLLQQFYSALAPFFFSLILDPASLQLLLNLLAQLIGVTPGGGATGPTGPTGPGGGATGPTGPTGPGGGATGPTGATGPTGDTGLAGATGATGPTGDTGVAGPAGPTGPTGDTGLAGATGPTGPTGDTGLAGATGPTGATGLAGATGPTGATGLTGATGATGAAGGGAIIPFASGTTPAALVNALIANTGTLLGFGFSQPGIGLAGGTSITLALGVGDYAFVAPRDGVITSLAGFFSATAALSPLSPVQVQIQILTAPAASNTFTVQGAPLLLTPAFAAIAIGSTASGIIPEAIPVVAGDKILLYVSLTAASPIAAVAGFVSAGINIV Full length YjcAMLFTSWLLFFIFALAAFRLTRLIVYDKITGFLRRPFIDELEITEPDGSVST (B. anthracis Ames)FTKVKGKGLRKWIGELLSCYWCTGVWVSAFLLVLYNWIPIVAEPLLA (SEQ ID NO: 80)LLAIAGAAAIIETITGYFMGE Full length YjcBMFAVSNNPRQNSYDLQQWYHMQQQHQAQQQAYQEQLQQQGFVKK (B. anthracis)KGCNCGKKKSTIKHYEE (SEQ ID NO: 81) Full length BclCMSRYDDSQNKFSKPCFPSSAGRIPNTPSIPVTKAQLRTFRAIIIDLTKIIP(B. anthracis Sterne)KLFANPSPQNIEDLIDTLNLLSKFICSLDAASSLKAQGLAIIKNLITILKN (SEQ ID NO: 82)PTFVASAVFIELQNLINYLLSITKLFRIDPCTLQELLKLIAALQTALVNSASFIQGPTGPTGPTGPTGPAGATGATGPQGVQGPAGATGATGPQGVQGPAGATGATGPQGAQGPAGATGATGPQGAQGPAGATGATGPQGIQGPAGATGATGPQGVQGPTGATGIGVTGPTGPSGGPAGATGPQGPQGNTGATGPQGIQGPAGATGATGPQGAQGPAGATGATGPQGVQGPTGATGIGVTGPTGPSGPSFPVATIVVTNNIQQTVLQFNNFIFNTAINVNNIIFNGTDTVTVINAGIYVISVSISTTAPGCAPLGVGISINGAVATDNFSSNLIGDSLSFTTIETLTAGANISVQSTLNEITIPATGNTNIRLTVFRIA Full length acidMKMKRGITTLLSVAVLSTSLVACSGITEKTVAKEEKVKLTDQQLMAD phosphataseLWYQTAGEMKALYYQGYNIGQLKLDAVLAKGTEKKPAIVLDLDETV (Bacillus thuringiensisLDNSPHQAMSVKTGKGYPYKWDDWINKAEAEALPGAIDFLKYTESK serovar konkukian str.GVDIYYISNRKTNQLDATIKNLERVGAPQATKEHILLQDPKEKGKEKR 97-27)RELVSQTHDIVLFFGDNLSDFTGFDGKSVKDRNQAVADSKAQFGEKFI (SEQ ID NO: 83)IFPNPMYGDWEGALYDYDFKKSDAEKDKIRRDNLKSFDTK Full length InhA2MKKKKKLKPLAVLTTAAVLSSTFAFGGHAAYAETPTSSLPIDEHLIPE (B. thuringiensisERLAEALKQRGVIDQSASQAETSKAVEKYVEKKKGENPGKEILTGDS HD74)LTQEASDFMKKVKDAKMRENEQAQQPEVGPVAGQGAALNPGKLNG (SEQ ID NO: 84)KVPTTSAKQEEYNGAVRKDKVLVLLVEFSDFKHNNIDQEPGYMYSKDFNREHYQKMLFGDEPFTLFDGSKINTFKQYYEEQSGGSYTVDGTVTEWLTVPGKASDYGADAGTGHDNKGPLGPKDLVKEALKAAVAKGINLADFDQYDQYDQNGNGNKNEPDGIIDHLMVVHAGVGQEAGGGKLKDDAIWSHRSKLGSKPYAIDGTKSSVSNWGGKMAAYDYTIEPEDGAVGVFAHEYGHDLGLPDEYDTKYSGQGEPVESWSIMSGGSWAGKIAGTEPTSFSPQNKEFFQKNMKGNWANILEVDYDKLSKGIGVATYVDQSTTKSKRPGIVRVNLPDKDIKNIESAFGKKFYYSTKGNDIHTTLETPVFDLTNAKDAKFDYKAFYELEAKYDFLDVYAIAEDGTKTRIDRMGEKDIKGGADTTDGKWVDKSYDLSQFKGKKVKLQFEYLTDIAVAYKGFALDNAALTVDGKVVFSDDAEGQPAMTLKGFTVSNGFEQKKHNYYVEWRNYAGSDTALQYARGPVFNTGMVVWYADQSFTDNWVGVHPGEGFLGVVDSHPEAIVGTLNGQPTVKSSTRYQIADAAFSFDQTPAWKVNSPTRGIFDYKGLPGVAKFDDSKQYINSVIPDAGRKLPKLGLKFEVVGQAEDKSA GAVWLHR AA = amino acids*B. anthracis Sterne strain BclA has 100% sequence identity with B.thuringiensis BclA. Thus, SEQ ID NOs: 1, 2, and 59 also represent aminoacids 1-41 of B. thuringiensis BclA, full length B. thuringiensis BclA,and amino acids 1-196 of B. thuringiensis BclA, respectively. Likewise,SEQ ID NO: 60 also represents a methionine residue plus amino acids20-35 of B. thuringiensis BclA. **B. mycoides hypothetical proteinTIGR03720 has 100% sequence identity with B. mycoides hypotheticalprotein WP003189234. Thus, SEQ ID NOs: 57 and 58 also represent aminoacids 1-136 of B. mycoides hypothetical protein WP003189234 and fulllength B. mycoides hypothetical protein WP003189234, respectively.

Bacillus is a genus of rod-shaped bacteria. The Bacillus cereus familyof bacteria includes the species Bacillus anthracis, Bacillus cereus,Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides,Bacillus samanii, Bacillus gaemokensis, and Bacillus weihenstephensis.Under stressful environmental conditions, Bacillus cereus familybacteria undergo sporulation and form oval endospores that can staydormant for extended periods of time. The outermost layer of theendospores is known as the exosporium and comprises a basal layersurrounded by an external nap of hair-like projections. Filaments on thehair-like nap are predominantly formed by the collagen-like glycoproteinBclA, while the basal layer is comprised of a number of differentproteins. Another collagen-related protein, BclB, is also present in theexosporium and exposed on endospores of Bacillus cereus family members.BclA, the major constituent of the surface nap, has been shown to beattached to the exosporium with its amino-terminus (N-terminus)positioned at the basal layer and its carboxy-terminus (C-terminus)extending outward from the spore.

It was previously discovered that certain sequences from the N-terminalregions of BclA and BclB could be used to target a peptide or protein tothe exosporium of a Bacillus cereus endospore (see U.S. PatentApplication Nos. 2010/0233124 and 2011/0281316, and Thompson et al.,Targeting of the BclA and BclB proteins to the Bacillus anthracis sporesurface, Molecular Microbiology 70(2):421-34 (2008), the entirety ofeach of which is hereby incorporated by reference). It was also foundthat the BetA/BAS3290 protein of Bacillus anthracis localized to theexosporium.

In particular, amino acids 20-35 of BclA from Bacillus anthracis Sternestrain have been found to be sufficient for targeting to the exosporium.A sequence alignment of amino acids 1-41 of BclA (SEQ ID NO: 1) with thecorresponding N-terminal regions of several other Bacillus cereus familyexosporium proteins and Bacillus cereus family proteins having relatedsequences is shown in FIG. 1. As can be seen from FIG. 1, there is aregion of high-homology among all of the proteins in the regioncorresponding to amino acids 20-41 of BclA. However, in these sequences,the amino acids corresponding to amino acids 36-41 of BclA containsecondary structure and are not necessary for fusion proteinlocalization to the exosporium. The conserved targeting sequence regionof BclA (amino acids 20-35 of SEQ ID NO: 1) is shown in bold in FIG. 1and corresponds to the minimal targeting sequence needed forlocalization to the exosporium. A more highly conserved region spanningamino acids 25-35 of BclA within the targeting sequence is underlined inthe sequences in FIG. 1, and is the recognition sequence forExsFA/BxpB/ExsFB and homologs, which direct and assemble the describedproteins on the surface of the exosporium The amino acid sequences ofSEQ ID NOs. 3, 5, and 7 in FIG. 1 are amino acids 1-33 of Bacillusanthracis Sterne strain BetA/BAS3290, a methionine followed by aminoacids 2-43 of Bacillus anthracis Sterne strain BAS4623, and amino acids1-34 of Bacillus anthracis Sterne strain BclB, respectively. (ForBAS4623, it was found that replacing the valine present at position 1 inthe native protein with a methionine resulted in better expression.) Ascan be seen from FIG. 1, each of these sequences contains a conservedregion corresponding to amino acids 20-35 of BclA (SEQ ID NO: 1; shownin bold), and a more highly conserved region corresponding to aminoacids 20-35 of BclA (underlined).

Additional proteins from Bacillus cereus family members also contain theconserved targeting region. In particular, in FIG. 1, SEQ ID NO: 9 isamino acids 1-30 of Bacillus anthracis Sterne strain BAS1882, SEQ ID NO:11 is amino acids 1-39 of the Bacillus weihenstephensis KBAB4 2280 geneproduct, SEQ ID NO: 13 is amino acids 1-39 of the Bacillusweihenstephensis KBAB4 3572 gene product, SEQ ID NO: 15 is amino acids1-49 of Bacillus cereus VD200 exosporium leader peptide, SEQ ID NO: 17is amino acids 1-33 of Bacillus cereus VD166 exosporium leader peptide,SEQ ID NO: 19 is amino acids 1-39 of Bacillus cereus VD200 hypotheticalprotein IKG 04663, SEQ ID NO: 21 is amino acids 1-39 of Bacillusweihenstephensis KBAB4 YVTN β-propeller protein, SEQ ID NO: 23 is aminoacids 1-30 of Bacillus weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2363, SEQ ID NO: 25 is amino acids 1-30 of Bacillusweihenstephensis KBAB4 hypothetical protein bcerkbab4_2131, SEQ ID NO:27 is amino acids 1-36 of Bacillus weihenstephensis KBAB4 triple helixrepeat containing collagen, SEQ ID NO: 29 is amino acids 1-39 ofBacillus mycoides 2048 hypothetical protein bmyco0001_21660, SEQ ID NO:31 is amino acids 1-30 of Bacillus mycoides 2048 hypothetical proteinbmyc0001_22540, SEQ ID NO: 33 is amino acids 1-21 of Bacillus mycoides2048 hypothetical protein bmyc0001_21510, SEQ ID NO: 35 is amino acids1-22 of Bacillus thuringiensis 35646 collagen triple helix repeatprotein, SEQ ID NO: 43 is amino acids 1-35 of Bacillus cereushypothetical protein WP_69652, SEQ ID NO: 45 is amino acids 1-41 ofBacillus cereus exosporium leader WP016117717, SEQ ID NO: 47 is aminoacids 1-49 of Bacillus cereus exosporium peptide WP002105192, SEQ ID NO:49 is amino acids 1-38 of Bacillus cereus hypothetical protein WP87353,SEQ ID NO: 51 is amino acids 1-39 of Bacillus cereus exosporium peptide02112369, SEQ ID NO: 53 is amino acids 1-39 of Bacillus cereusexosporium protein WP016099770, SEQ ID NO: 55 is amino acids 1-36 ofBacillus thuringiensis hypothetical protein YP006612525, and SEQ ID NO:57 is amino acids 1-136 of Bacillus mycoides hypothetical proteinTIGR03720. As shown in FIG. 1, each of the N-terminal regions of theseproteins contains a region that is conserved with amino acids 20-35 ofBclA (SEQ ID NO: 1), and a more highly conserved region corresponding toamino acids 25-35 of BclA.

In the fusion proteins of the present invention, any portion of BclAwhich includes amino acids 20-35 can be used as the targeting sequencein the present invention. In addition, full-length exosporium proteinsor exosporium protein fragments can be used for targeting the fusionproteins to the exosporium. Thus, full-length BclA or a fragment of BclAthat includes amino acids 20-35 can be used for targeting to theexosporium. For example, full length BclA (SEQ ID NO: 2) or a midsizedfragment of BclA that lacks the carboxy-terminus such as SEQ ID NO: 59(amino acids 1-196 of BclA) can be used to target the fusion proteins tothe exosporium. Midsized fragments such as the fragment of SEQ ID NO: 59have less secondary structure than full length BclA and has been foundto be suitable for use as a targeting sequence. The targeting sequencecan also comprise much shorter portions of BclA which include aminoacids 20-35, such as SEQ ID NO: 1 (amino acids 1-41 of BclA), aminoacids 1-35 of SEQ ID NO: 1, amino acids 20-35 of SEQ ID NO: 1, or SEQ IDNO: 60 (a methionine residue linked to amino acids 20-35 of BclA). Evenshorter fragments of BclA which include only some of amino acids 20-35also exhibit the ability to target fusion proteins to the exosporium.For example, the targeting sequence can comprise amino acids 22-31 ofSEQ ID NO: 1, amino acids 22-33 of SEQ ID NO: 1, or amino acids 20-31 ofSEQ ID NO: 1.

Alternatively, any portion of BetA/BAS3290, BAS4623, BclB, BAS1882, theKBAB4 2280 gene product, the KBAB4 3572 gene product, B. cereus VD200exosporium leader peptide, B. cereus VD166 exosporium leader peptide, B.cereus VD200 hypothetical protein IKG 04663, B. weihenstephensis KBAB4YVTN β-propeller protein, B. weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2363, B. weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2131, B. weihenstephensis KBAB4 triple helix repeat containingcollagen, B. mycoides 2048 hypothetical protein bmyco0001_21660, B.mycoides 2048 hypothetical protein bmyc0001_22540, B. mycoides 2048hypothetical protein bmyc0001_21510, B. thuringiensis 35646 collagentriple helix repeat protein, B. cereus hypothetical protein WP_69652, B.cereus exosporium leader WP016117717, B. cereus exosporium peptideWP002105192, B. cereus hypothetical protein WP87353, B. cereusexosporium peptide 02112369, B. cereus exosporium protein WP016099770,B. thuringiensis hypothetical protein YP006612525, or B. mycoideshypothetical protein TIGR03720 which includes the amino acidscorresponding to amino acids 20-35 of BclA can serve as the targetingsequence. As can be seen from FIG. 1, amino acids 12-27 of BetA/BAS3290,amino acids 23-38 of BAS4623, amino acids 13-28 of BclB, amino acids9-24 of BAS1882, amino acids 18-33 of KBAB4 2280 gene product, aminoacids 18-33 of KBAB4 3572 gene product, amino acids 28-43 of B. cereusVD200 exosporium leader peptide, amino acids 12-27 of B. cereus VD166exosporium leader peptide, amino acids 18-33 of B. cereus VD200hypothetical protein IKG_04663, amino acids 18-33 B. weihenstephensisKBAB4 YVTN β-propeller protein, amino acids 9-24 of B. weihenstephensisKBAB4 hypothetical protein bcerkbab4_2363, amino acids 9-24 of B.weihenstephensis KBAB4 hypothetical protein bcerkbab4_2131, amino acids15-30 of B. weihenstephensis KBAB4 triple helix repeat containingcollagen, amino acids 18-33 of B. mycoides 2048 hypothetical proteinbmyco0001_21660, amino acids 9-24 of B. mycoides 2048 hypotheticalprotein bmyc0001_22540, amino acids 1-15 of B. mycoides 2048hypothetical protein bmyc0001_21510, amino acids 1-16 of B.thuringiensis 35646 collagen triple helix repeat protein, amino acids14-29 of B. cereus hypothetical protein WP_69652, amino acids 20-35 ofB. cereus exosporium leader WP016117717, amino acids 28-43 of B. cereusexosporium peptide WP002105192, amino acids 17-32 of B. cereushypothetical protein WP87353, amino acids 18-33 of B. cereus exosporiumpeptide 02112369, amino acids 18-33 of B. cereus exosporium proteinWP016099770, amino acids 15-30 of B. thuringiensis hypothetical proteinYP006612525, and amino acids 115-130 of B. mycoides hypothetical proteinTIGR03720 correspond to amino acids 20-35 of BclA. Thus, any portion ofthese proteins that includes the above-listed corresponding amino acidscan serve as the targeting sequence.

Furthermore, any amino acid sequence comprising amino acids 20-35 ofBclA, or any of the above-listed corresponding amino acids can serve asthe targeting sequence.

Thus, the targeting sequence can comprise amino acids 1-35 of SEQ ID NO:1, amino acids 20-35 of SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 60, aminoacids 22-31 of SEQ ID NO: 1, amino acids 22-33 of SEQ ID NO: 1, or aminoacids 20-31 of SEQ ID NO: 1. Alternatively, the targeting sequenceconsists of amino acids 1-35 of SEQ ID NO: 1, amino acids 20-35 of SEQID NO: 1, SEQ ID NO: 1, or SEQ ID NO: 60. Alternatively, the targetingsequence can consist of amino acids 22-31 of SEQ ID NO: 1, amino acids22-33 of SEQ ID NO: 1, or amino acids 20-31 of SEQ ID NO: 1.Alternatively, the exosporium protein can comprise full length BclA (SEQID NO: 2), or the exosporium protein fragment can comprise a midsizedfragment of BclA that lacks the carboxy-terminus, such as SEQ ID NO: 59(amino acids 1-196 of BclA). Alternatively, the exosporium proteinfragment can consist of SEQ ID NO: 59.

The targeting sequence can also comprise amino acids 1-27 of SEQ ID NO:3, amino acids 12-27 of SEQ ID NO: 3, or SEQ ID NO: 3, or the exosporiumprotein can comprise full length BetA/BAS3290 (SEQ ID NO: 4). It hasalso been found that a methionine residue linked to amino acids 12-27 ofBetA/BAS3290 can be used as a targeting sequence. Thus, the targetingsequence can comprise SEQ ID NO: 61. The targeting sequence can alsocomprise amino acids 14-23 of SEQ ID NO: 3, amino acids 14-25 of SEQ IDNO: 3, or amino acids 12-23 of SEQ ID NO: 3.

The targeting sequence can also comprise amino acids 1-38 of SEQ ID NO:5, amino acids 23-38 of SEQ ID NO: 5, or SEQ ID NO: 5, or the exosporiumprotein can comprise full length BAS4623 (SEQ ID NO: 6).

Alternatively, the targeting sequence can comprise amino acids 1-28 ofSEQ ID NO: 7, amino acids 13-28 of SEQ ID NO: 7, or SEQ ID NO: 7, or theexosporium protein can comprise full length BclB (SEQ ID NO:8).

The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO:9, amino acids 9-24 of SEQ ID NO: 9, or SEQ ID NO: 9, or the exosporiumprotein can comprise full length BAS1882 (SEQ ID NO: 10). A methionineresidue linked to amino acids 9-24 of BAS1882 can also be used as atargeting sequence. Thus, the targeting sequence can comprise SEQ ID NO:69.

The targeting sequence can also comprise amino acids 1-33 of SEQ IDNO:11, amino acids 18-33 of SEQ ID NO: 11, or SEQ ID NO: 11, or theexosporium protein can comprise the full length B. weihenstephensisKBAB4 2280 gene product (SEQ ID NO: 12). A methionine residue linked toamino acids 18-33 of the B. weihenstephensis KBAB4 2280 gene product canalso be used as a targeting sequence. Thus, the targeting sequence cancomprise SEQ ID NO: 62.

The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO:13, amino acids 18-33 of SEQ ID NO: 13, or SEQ ID NO:13, or theexosporium protein can comprise the full length B. weihenstephensisKBAB4 3572 gene product (SEQ ID NO:14). A methionine residue linked toamino acids 18-33 of the B. weihenstephensis KBAB4 3572 gene product canalso be used as a targeting sequence. Thus, the targeting sequence cancomprise SEQ ID NO: 63.

Alternatively, the targeting sequence can comprise amino acids 1-43 ofSEQ ID NO: 15, amino acids 28-43 of SEQ ID NO: 15, or SEQ ID NO:15, orthe exosporium protein can comprise full length B. cereus VD200exosporium leader peptide (SEQ ID NO:16).

The targeting sequence can also comprise amino acids 1-27 of SEQ ID NO:17, amino acids 12-27 of SEQ ID NO: 17, or SEQ ID NO:17, or theexosporium protein can comprise full-length B. cereus VD166 exosporiumleader peptide (SEQ ID NO:18). A methionine residue linked to aminoacids 12-27 of the B. cereus VD166 exosporium leader peptide can also beused as a targeting sequence. Thus, the targeting sequence can compriseSEQ ID NO: 64.

The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO:19, amino acids 18-33 of SEQ ID NO: 19, or SEQ ID NO:19, or theexosporium protein can comprise full length B. cereus VD200 hypotheticalprotein IKG 04663 (SEQ ID NO:20).

Alternatively, the targeting sequence comprises amino acids 1-33 of SEQID NO: 21, amino acids 18-33 of SEQ ID NO: 21, or SEQ ID NO:21, or theexosporium protein can comprise full length B. weihenstephensis KBAB4YVTN β-propeller protein (SEQ ID NO:22). A methionine residue linked toamino acids 18-33 of the B. weihenstephensis KBAB4 YVTN β-propellerprotein can also be used as a targeting sequence. Thus, the targetingsequence can comprise SEQ ID NO: 65.

The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO:23, amino acids 9-24 of SEQ ID NO: 23, or SEQ ID NO:23, or theexosporium protein can comprise full length B. weihenstephensis KBAB4hypothetical protein bcerkbab4_2363 (SEQ ID NO:24). A methionine residuelinked to amino acids 9-24 of B. weihenstephensis KBAB4 hypotheticalprotein bcerkbab4_2363 can also be used as a targeting sequence. Thus,the targeting sequence can comprise SEQ ID NO: 66.

The targeting sequence comprise amino acids 1-24 of SEQ ID NO: 25, aminoacids 9-24 of SEQ ID NO: 25, or SEQ ID NO:25, or the exosporium proteincan comprise full length B. weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2131 (SEQ ID NO:26). A methionine residue linked to aminoacids 9-24 of B. weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2131 can also be used as a targeting sequence. Thus, thetargeting sequence can comprise SEQ ID NO: 67.

Alternatively, the targeting sequence comprises amino acids 1-30 of SEQID NO: 27, amino acids 15-30 of SEQ ID NO: 27, or SEQ ID NO:27, or theexosporium protein can comprise full length B. weihenstephensis KBAB4triple helix repeat containing collagen (SEQ ID NO:28).

The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO:29, amino acids 18-33 of SEQ ID NO: 29, or SEQ ID NO:29, or theexosporium protein can comprise full length B. mycoides 2048hypothetical protein bmyco0001_21660 (SEQ ID NO:30).

The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO:31, amino acids 9-24 of SEQ ID NO: 31, or SEQ ID NO:31, or theexosporium protein can comprise full length B. mycoides 2048hypothetical protein bmyc0001_22540 (SEQ ID NO:32). A methionine residuelinked to amino acids 9-24 of B. mycoides 2048 hypothetical proteinbmyc0001_22540 can also be used as a targeting sequence. Thus, thetargeting sequence can comprise SEQ ID NO: 68.

Alternatively, the targeting sequence comprises amino acids 1-15 of SEQID NO: 33, SEQ ID NO:33, or the exosporium protein comprises full lengthB. mycoides 2048 hypothetical protein bmyc0001_21510 (SEQ ID NO:34).

The targeting sequence can also comprise amino acids 1-16 of SEQ ID NO:35, SEQ ID NO:35, or the exosporium protein can comprise full length B.thuringiensis 35646 collagen triple helix repeat protein (SEQ ID NO:36).

The targeting sequence can comprise amino acids 1-29 of SEQ ID NO:43,amino acids 14-29 of SEQ ID NO: 43, or SEQ ID NO: 43, or the exosporiumprotein can comprise full length B. cereus hypothetical protein WP_69652(SEQ ID NO: 44).

Alternatively, the targeting sequence can comprise amino acids 1-35 ofSEQ ID NO: 45, amino acids 20-35 of SEQ ID NO: 45, or SEQ ID NO: 45, orthe exosporium protein can comprise full length B. cereus exosporiumleader WP016117717 (SEQ ID NO: 46). A methionine residue linked to aminoacids 20-35 of B. cereus exosporium leader WP016117717 can also be usedas a targeting sequence. Thus, the targeting sequence can comprise SEQID NO: 70.

The targeting sequence can comprise amino acids 1-43 of SEQ ID NO: 47,amino acids 28-43 of SEQ ID NO: 47, or SEQ ID NO: 47, or the exosporiumprotein can comprise full length B. cereus exosporium peptideWP002105192 (SEQ ID NO: 48).

The targeting sequence can comprise amino acids 1-32 of SEQ ID NO: 49,amino acids 17-32 of SEQ ID NO: 49, or SEQ ID NO: 49, or the exosporiumprotein can comprise full length B. cereus hypothetical protein WP87353(SEQ ID NO: 50).

Alternatively, the targeting sequence can comprise amino acids 1-33 ofSEQ ID NO: 51, amino acids 18-33 of SEQ ID NO: 51, or SEQ ID NO: 51, orthe exosporium protein can comprise full length B. cereus exosporiumpeptide 02112369 (SEQ ID NO: 52).

The targeting sequence can comprise amino acids 1-33 of SEQ ID NO: 53,amino acids 18-33 of SEQ ID NO: 53, or SEQ ID NO: 53, or the exosporiumprotein can comprise full length B. cereus exosporium proteinWP016099770 (SEQ ID NO: 54).

Alternatively, the targeting sequence can comprise acids 1-30 of SEQ IDNO: 55, amino acids 15-30 of SEQ ID NO: 55, or SEQ ID NO: 55, or theexosporium protein can comprise full length B. thuringiensishypothetical protein YP006612525 (SEQ ID NO: 56).

The targeting sequence can also comprise amino acids 1-130 of SEQ ID NO:57, amino acids 115-130 of SEQ ID NO: 57, or SEQ ID NO: 57, or theexosporium protein can comprise full length B. mycoides hypotheticalprotein TIGR03720 (SEQ ID NO: 58).

In addition, it can readily be seen from the sequence alignment in FIG.1 that while amino acids 20-35 of BclA are conserved, and amino acids25-35 are more conserved, some degree of variation can occur in thisregion without affecting the ability of the targeting sequence to targeta protein to the exosporium. FIG. 1 lists the percent identity of eachof corresponding amino acids of each sequence to amino acids 20-35 ofBclA (“20-35% Identity”) and to amino acids 25-35 of BclA (“25-35%Identity”). Thus, for example, as compared to amino acids 20-35 of BclA,the corresponding amino acids of BetA/BAS3290 are about 81.3% identical,the corresponding amino acids of BAS4623 are about 50.0% identical, thecorresponding amino acids of BclB are about 43.8% identical, thecorresponding amino acids of BAS1882 are about 62.5% identical, thecorresponding amino acids of the KBAB4 2280 gene product are about 81.3%identical, and the corresponding amino acids of the KBAB4 3572 geneproduct are about 81.3% identical. The sequence identities over thisregion for the remaining sequences are listed in FIG. 1.

With respect to amino acids 25-35 of BclA, the corresponding amino acidsof BetA/BAS3290 are about 90.9% identical, the corresponding amino acidsof BAS4623 are about 72.7% identical, the corresponding amino acids ofBclB are about 54.5% identical, the corresponding amino acids of BAS1882are about 72.7% identical, the corresponding amino acids of the KBAB42280 gene product are about 90.9% identical, and the corresponding aminoacids of the KBAB4 3572 gene product are about 81.8% identical. Thesequence identities over this region for the remaining sequences arelisted in FIG. 1.

Thus, the targeting sequence can comprise an amino acid sequence havingat least about 43% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 54%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 43% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 54%.

The targeting sequence can also comprise an amino acid sequence havingat least about 50% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 63%.Alternatively the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 50% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 63%.

The targeting sequence can also comprise an amino acid sequence havingat least about 50% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 72%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 50% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 72%.

The targeting sequence can also comprise an amino acid sequence havingat least about 56% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 63%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 56% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 63%.

Alternatively, the targeting sequence can comprise an amino sequencehaving at least about 62% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 72%.The targeting sequence can also consist of an amino acid sequenceconsisting of 16 amino acids and having at least about 62% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 of SEQ ID NO:1 is at least about 72%.

The targeting sequence can comprise an amino acid sequence having atleast 68% identity with amino acids 20-35 of SEQ ID NO: 1, wherein theidentity with amino acids 25-35 is at least about 81%. Alternatively,the targeting sequence consists of an amino acid sequence consisting of16 amino acids and having at least 68% identity with amino acids 20-35of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at leastabout 81%.

The targeting sequence can also comprises an amino sequence having atleast about 75% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 72%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 75% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 of SEQ ID NO:1 is at least about 72%.

The targeting sequence can also comprise an amino sequence having atleast about 75% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 81%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 75% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 of SEQ ID NO:1 is at least about 81%.

The targeting sequence can also comprise an amino acid sequence havingat least about 81% identity with amino acids 20-35 of SEQ ID NO:1,wherein the identity with amino acids 25-35 is at least about 81%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 81% identity withamino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids25-35 is at least about 81%.

The targeting sequence can comprise an amino acid sequence having atleast about 81% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 90%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 81% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 90%.

The skilled person will recognize that variants of the above sequencescan also be used as targeting sequences, so long as the targetingsequence comprises amino acids 20-35 of BclA, the corresponding aminoacids of BetA/BAS3290, BAS4263, BclB, BAS1882, the KBAB4 2280 geneproduct, or the KBAB 3572 gene product, or a sequence comprising any ofthe above noted sequence identities to amino acids 20-35 and 25-35 ofBclA is present.

It has further been discovered that certain Bacillus cereus familyexosporium proteins which lack regions having homology to amino acids25-35 of BclA can also be used to target a peptide or protein to theexosporium of a Bacillus cereus family member. In particular, the fusionproteins can comprise an exosporium protein comprising SEQ ID NO: 71 (B.mycoides InhA), an exosporium protein comprising SEQ ID NO: 72 (B.anthracis Sterne BAS1141 (ExsY)), an exosporium protein comprising SEQID NO: 73 (B. anthracis Sterne BAS1144 (BxpB/ExsFA)), an exosporiumprotein comprising SEQ ID NO: 74 (B. anthracis Sterne BAS1145 (CotY)),an exosporium protein comprising SEQ ID NO: 75 (B. anthracis SterneBAS1140), an exosporium protein comprising SEQ ID NO: 76 (B. anthracisH9401 ExsFB), an exosporium protein comprising SEQ ID NO: 77 (B.thuringiensis HD74 InhA1), an exosporium protein comprising SEQ ID NO:78 (B. cereus ATCC 10876 ExsJ), an exosporium protein comprising SEQ IDNO: 79 (B. cereus ExsH), an exosporium protein comprising SEQ ID NO: 80(B. anthracis Ames YjcA), an exosporium protein comprising SEQ ID NO: 81(B. anthracis YjcB), an exosporium protein comprising SEQ ID NO: 82 (B.anthracis Sterne BclC), an exosporium protein comprising SEQ ID NO: 83(Bacillus thuringiensis serovar konkukian str. 97-27 acid phosphatase),or an exosporium protein comprising SEQ ID NO: 84 (B. thuringiensis HD74InhA2). Inclusion of an exosporium protein comprising SEQ ID NO: 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 in the fusion proteinsdescribed herein will result in targeting to the exosporium of a B.cereus family member.

Moreover, exosporium proteins having a high degree of sequence identitywith any of the full-length exosporium proteins or the exosporiumprotein fragments described above can also be used to target a peptideor protein to the exosporium of a Bacillus cereus family member. Thus,the fusion protein can comprise an exosporium protein comprising anamino acid sequence having at least 85% identity with any one of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,44, 46, 48, 50, 52, 54, 56, 58, 59, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, and 84. Alternatively, the fusion protein can comprisean exosporium protein having at least 90%, at least 95%, at least 98%,at least 99%, or 100% identity with any one of SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50,52, 54, 56, 58, 59, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,and 84.

Alternatively, the fusion protein can comprise an exosporium proteinfragment consisting of an amino acid sequence having at least 85%identity with SEQ ID NO: 59. Alternatively, the fusion protein cancomprise an exosporium protein fragment consisting of an amino acidsequence having at least 90%, at least 95%, at least 98%, at least 99%,or 100% identity with SEQ ID NO: 59.

In any of the targeting sequences, exosporium proteins, or exosporiumprotein fragments described herein, the targeting sequence, exosporiumprotein, or exosporium protein fragment can comprise the amino acidsequence GXT at its carboxy terminus, wherein X is any amino acid.

In any of the targeting sequences, exosporium proteins, and exosporiumprotein fragments described herein, the targeting sequence, exosporiumprotein, or exosporium protein fragment, can comprise an alanine residueat the position of the targeting sequence that corresponds to amino acid20 of SEQ ID NO: 1.

Fusion Proteins

The present invention relates to fusion proteins comprising a targetingsequence, an exosporium protein, or an exosporium protein fragment, andat least one plant growth stimulating protein or peptide, wherein theplant growth stimulating protein or peptide comprises a peptide hormone,a non-hormone peptide, or an enzyme involved in the production oractivation of a plant growth stimulating compound. The targetingsequence, exosporium protein, or exosporium protein fragment can be anyof the targeting sequences, exosporium proteins, or exosporium proteinfragments described above in paragraph [0005].

The present invention additionally relates to fusion proteins comprisinga targeting sequence, an exosporium protein, or an exosporium proteinfragment, and at least one protein or peptide that enhances stressresistance in a plant. The targeting sequence, exosporium protein, orexosporium protein fragment can be any of the targeting sequences,exosporium proteins, or exosporium protein fragments described above inparagraph [0005].

In addition, the present invention relates to fusion proteins comprisinga targeting sequence, an exosporium protein, or an exosporium proteinfragment, and at least one plant binding protein or peptide. Thetargeting sequence, exosporium protein, or exosporium protein fragmentcan be any of the targeting sequences, exosporium proteins, orexosporium protein fragments described above in paragraph [0005].

The present invention also relates to fusion proteins comprising atargeting sequence, an exosporium protein, or an exosporium proteinfragment, and at least one protein or peptide that protects a plant froma pathogen. The targeting sequence, exosporium protein, or exosporiumprotein fragment can be any of the targeting sequences, exosporiumproteins, or exosporium protein fragments described above in paragraph[0006].

The present invention further relates to fusion proteins comprising atargeting sequence, an exosporium protein, or an exosporium proteinfragment, and at least one protein or peptide that protects a plant froma pathogen. The protein or peptide that protects a plant from a pathogencomprises a harpin, an α-elastin, a β-elastin, a systemin, aphenylalanine ammonia-lyase, an elicitin, a defensin, a cryptogein, aflagellin protein, a flagellin peptide, a bacteriocin, a lysozyme, alysozyme peptide, a siderophore, a non-ribosomal active peptide, aconalbumin, an albumin, a lactoferrin, a lactoferrin peptide, TasA.Alternatively, the protein or peptide that protects a plant from apathogen has insecticidal activity, helminthicidal activity, suppressesinsect or worm predation, or a combination thereof. Alternatively, theprotein that protects a plant from a pathogen comprises an enzyme. Thetargeting sequence, exosporium protein, or exosporium protein fragmentcan be any of the targeting sequences, exosporium proteins, orexosporium protein fragments described above in paragraph [0005].

The fusion protein can be made using standard cloning and molecularbiology methods known in the art. For example, a gene encoding a proteinor peptide (e.g., a gene encoding a plant growth stimulating protein orpeptide) can be amplified by polymerase chain reaction (PCR) and ligatedto DNA coding for any of the above-described targeting sequences to forma DNA molecule that encodes the fusion protein. The DNA moleculeencoding the fusion protein can be cloned into any suitable vector, forexample a plasmid vector. The vector suitably comprises a multiplecloning site into which the DNA molecule encoding the fusion protein canbe easily inserted. The vector also suitably contains a selectablemarker, such as an antibiotic resistance gene, such that bacteriatransformed, transfected, or mated with the vector can be readilyidentified and isolated. Where the vector is a plasmid, the plasmidsuitably also comprises an origin of replication. The DNA encoding thefusion protein is suitably under the control of a sporulation promoterwhich will cause expression of the fusion protein on the exosporium of aB. cereus family member endospore (e.g., a native bclA promoter from aB. cereus family member). Alternatively, DNA coding for the fusionprotein can be integrated into the chromosomal DNA of the B. cereusfamily member host.

The fusion protein can also comprise additional polypeptide sequencesthat are not part of the targeting sequence, exosporium protein,exosporium protein fragment, or the plant growth stimulating protein orpeptide, the protein or peptide that protects a plant from a pathogen,the protein or peptide that enhances stress resistance in a plant, orthe plant binding protein or peptide. For example, the fusion proteincan include tags or markers to facilitate purification or visualizationof the fusion protein (e.g., a polyhistidine tag or a fluorescentprotein such as GFP or YFP) or visualization of recombinant Bacilluscereus family member spores expressing the fusion protein.

Expression of fusion proteins on the exosporium using the targetingsequences, exosporium proteins, and exosporium protein fragmentsdescribed herein is enhanced due to a lack of secondary structure in theamino-termini of these sequences, which allows for native folding of thefused proteins and retention of activity. Proper folding can be furtherenhanced by the inclusion of a short amino acid linker between thetargeting sequence, exosporium protein, exosporium protein fragment, andthe fusion partner protein.

Thus, any of the fusion proteins described herein can comprise an aminoacid linker between the targeting sequence, the exosporium protein, orthe exosporium protein fragment and the plant growth stimulating proteinor peptide, the protein or peptide that protects a plant from apathogen, the protein or peptide that enhances stress resistance in aplant, or the plant binding protein or peptide.

The linker can comprise a polyalanine linker or a polyglycine linker. Alinker comprising a mixture of both alanine and glycine residues canalso be used. For example, where the targeting sequence comprises SEQ IDNO: 1, a fusion protein can have one of the following structures:

No linker: SEQ ID NO: 1—Fusion Partner Protein

Alanine Linker: SEQ ID NO: 1—A_(n)—Fusion Partner Protein

Glycine Linker: SEQ ID NO: 1—G_(n)—Fusion Partner Protein

Mixed Alanine and Glycine Linker: SEQ ID NO: 1—(A/G)_(n)—Fusion PartnerProtein where A_(n), G_(n), and (A/G)_(n) are any number of alanines,any number of glycines, or any number of a mixture of alanines andglycines, respectively. For example, n can be 1 to 25, and is preferably6 to 10. Where the linker comprises a mixture of alanine and glycineresidues, any combination of glycine and alanine residues can be used.In the above structures, “Fusion Partner Protein” represents the plantgrowth stimulating protein or peptide, the protein or peptide thatprotects a plant from a pathogen, the protein or peptide that enhancesstress resistance in a plant, or the plant binding protein or peptide.

Alternatively or in addition, the linker can comprise a proteaserecognition site. Inclusion of a protease recognition site allows fortargeted removal, upon exposure to a protease that recognizes theprotease recognition site, of the plant growth stimulating protein orpeptide, the protein or peptide that protects a plant from a pathogen,the protein or peptide that enhances stress resistance in a plant, orthe plant binding protein or peptide.

Plant Growth Stimulating Proteins and Peptides

As noted above, the present invention relates to fusion proteinscomprising a targeting sequence, exosporium protein, or exosporiumprotein fragment and at least one plant growth stimulating protein orpeptide, wherein the plant growth stimulating protein or peptidecomprises a peptide hormone, a non-hormone peptide, or an enzymeinvolved in the production or activation of a plant growth stimulatingcompound.

For example, where the plant growth stimulating protein or peptidecomprises a peptide hormone, the peptide hormone can comprise aphytosulfokine (e.g., phytosulfokine-α), clavata 3 (CLV3), systemin,Zm1GF, or a SCR/SP11.

Where the plant growth stimulating protein or peptide comprises anon-hormone peptide, the non-hormone peptide can comprise a RKN 16D10,Hg-Syv46, an eNOD40 peptide, melittin, mastoparan, Mas7, RHPP, POLARIS,or kunitz trypsin inhibitor (KTI).

The plant growth stimulating protein or peptide can comprise an enzymeinvolved in the production or activation of a plant growth stimulatingcompound. The enzyme involved in the production or activation of a plantgrowth stimulating compound can be any enzyme that catalyzes any step ina biological synthesis pathway for a compound that stimulates plantgrowth or alters plant structure, or any enzyme that catalyzes theconversion of an inactive or less active derivative of a compound thatstimulates plant growth or alters plant structure into an active or moreactive form of the compound.

The plant growth stimulating compound can comprise a compound producedby bacteria or fungi in the rhizosphere, e.g., 2,3-butanediol.

Alternatively, the plant growth stimulating compound can comprise aplant growth hormone, e.g., a cytokinin or a cytokinin derivative,ethylene, an auxin or an auxin derivative, a gibberellic acid or agibberellic acid derivative, abscisic acid or an abscisic acidderivative, or a jasmonic acid or a jasmonic acid derivative.

Where the plant growth stimulating compound comprises a cytokinin or acytokinin derivative, the cytokinin or the cytokinin derivative cancomprise kinetin, cis-zeatin, trans-zeatin, 6-benzylaminopurine,dihydroxyzeatin, N6-(D2-isopentenyl) adenine, ribosylzeatin,N6-(D2-isopentenyl) adenosine, 2-methylthio-cis-ribosylzeatin,cis-ribosylzeatin, trans-ribosylzeatin,2-methylthio-trans-ribosylzeatin, ribosylzeatin-5-monosphosphate,N6-methylaminopurine, N6-dimethylaminopurine, 2′-deoxyzeatin riboside,4-hydroxy-3-methyl-trans-2-butenylaminopurine, ortho-topolin,meta-topolin, benzyladenine, ortho-methyltopolin, meta-methyltopolin, ora combination thereof.

Where the plant growth stimulating compound comprises an auxin or anauxin derivative, the auxin or the auxin derivative can comprise anactive auxin, an inactive auxin, a conjugated auxin, a naturallyoccurring auxin, or a synthetic auxin, or a combination thereof. Forexample, the auxin or auxin derivative can comprise indole-3-aceticacid, indole-3-pyruvic acid, indole-3-acetaldoxime, indole-3-acetamide,indole-3-acetonitrile, indole-3-ethanol, indole-3-pyruvate,indole-3-acetaldoxime, indole-3-butyric acid, a phenylacetic acid,4-chloroindole-3-acetic acid, a glucose-conjugated auxin, or acombination thereof.

The enzyme involved in the production or activation of a plant growthstimulating compound can comprise an acetoin reductase, anindole-3-acetamide hydrolase, a tryptophan monooxygenase, anacetolactate synthetase, an α-acetolactate decarboxylase, a pyruvatedecarboxylase, a diacetyl reductase, a butanediol dehydrogenase, anaminotransferase (e.g., tryptophan aminotransferase), a tryptophandecarboxylase, an amine oxidase, an indole-3-pyruvate decarboxylase, anindole-3-acetaldehyde dehydrogenase, a tryptophan side chain oxidase, anitrile hydrolase, a nitrilase, a peptidase, a protease, an adenosinephosphate isopentenyltransferase, a phosphatase, an adenosine kinase, anadenine phosphoribosyltransferase, CYP735A, a 5′ ribonucleotidephosphohydrolase, an adenosine nucleosidase, a zeatin cis-transisomerase, a zeatin O-glucosyltransferase, a β-glucosidase, acis-hydroxylase, a CK cis-hydroxylase, a CK N-glucosyltransferase, a2,5-ribonucleotide phosphohydrolase, an adenosine nucleosidase, a purinenucleoside phosphorylase, a zeatin reductase, a hydroxylamine reductase,a 2-oxoglutarate dioxygenase, a gibberellic 2B/3B hydrolase, agibberellin 3-oxidase, a gibberellin 20-oxidase, a chitosinase, achitinase, a β-1,3-glucanase, a β-1,4-glucanase, a β-1,6-glucanase, anaminocyclopropane-1-carboxylic acid deaminase, or an enzyme involved inproducing a nod factor (e.g., nodA, nodB, or nodI).

Where the enzyme comprises a protease or peptidase, the protease orpeptidase can be a protease or peptidase that cleaves proteins,peptides, proproteins, or preproproteins to create a bioactive peptide.The bioactive peptide can be any peptide that exerts a biologicalactivity.

Examples of bioactive peptides include RKN 16D10 and RHPP.

The protease or peptidase that cleaves proteins, peptides, proproteins,or preproproteins to create a bioactive peptide can comprise subtilisin,an acid protease, an alkaline protease, a proteinase, an endopeptidase,an exopeptidase, thermolysin, papain, pepsin, trypsin, pronase, acarboxylase, a serine protease, a glutamic protease, an aspartateprotease, a cysteine protease, a threonine protease, or ametalloprotease.

The protease or peptidase can cleave proteins in a protein-rich meal(e.g., soybean meal or yeast extract).

Proteins and Peptides that Protects Plants from Pathogens

The present invention relates to fusion proteins comprising a targetingsequence, exosporium protein, or exosporium protein fragment, and atleast one protein or peptide that protects a plant from a pathogen.

The protein or peptide that protects a plant from a pathogen cancomprise a protein or peptide that stimulates a plant immune response.For example, the protein or peptide that stimulates a plant immuneresponse can comprise a plant immune system enhancer protein or peptide.The plant immune system enhancer protein or peptide can be any proteinor peptide that has a beneficial effect on the immune system of a plant.Suitable plant immune system enhancer proteins and peptides includeharpins, α-elastins, β-elastins, systemins, phenylalanine ammonia-lyase,elicitins, defensins, cryptogeins, flagellin proteins, and flagellinpeptides (e.g., flg22).

Alternatively, the protein or peptide that protects a plant from apathogen can be a protein or peptide that has antibacterial activity,antifungal activity, or both antibacterial and antifungal activity.Examples of such proteins and peptides include bacteriocins, lysozymes,lysozyme peptides (e.g., LysM), siderophores, non-ribosomal activepeptides, conalbumins, albumins, lactoferrins, lactoferrin peptides(e.g., LfcinB), and TasA.

The protein or peptide that protects a plant from a pathogen can also bea protein or peptide that has insecticidal activity, helminthicidalactivity, suppresses insect or worm predation, or a combination thereof.For example, the protein or peptide that protects a plant from apathogen can comprise an insecticidal bacterial toxin (e.g., a VIPinsecticidal protein), an endotoxin, a Cry toxin (e.g., a Cry toxin fromBacillus thuringiensis), a protease inhibitor protein or peptide (e.g.,a trypsin inhibitor or an arrowhead protease inhibitor), a cysteineprotease, or a chitinase. Where the Cry toxin is a Cry toxin fromBacillus thuringiensis, the Cry toxin can be a Cry5B protein or a Cry21Aprotein. Cry5B and Cry21A have both insecticidal and nematocidalactivity.

The protein that protects a plant from a pathogen can comprise anenzyme. Suitable enzymes include proteases and lactonases. The proteasesand lactonases can be specific for a bacterial signaling molecule (e.g.,a bacterial lactone homoserine signaling molecule).

Where the enzyme is a lactonase, the lactonase can comprise1,4-lactonase, 2-pyrone-4,6-dicarboxylate lactonase, 3-oxoadipateenol-lactonase, actinomycin lactonase, deoxylimonate A-ring-lactonase,gluconolactonase L-rhamnono-1,4-lactonase, limonin-D-ring-lactonase,steroid-lactonase, triacetate-lactonase, or xylono-1,4-lactonase.

The enzyme can also be an enzyme that is specific for a cellularcomponent of a bacterium or fungus. For example, the enzyme can comprisea β-1,3-glucanase, a β-1,4-glucanase, a β-1,6-glucanase, a chitosinase,a chitinase, a chitosinase-like enzyme, a lyticase, a peptidase, aproteinase, a protease (e.g., an alkaline protease, an acid protease, ora neutral protease), a mutanolysin, a stapholysin, or a lysozyme.

For any of the above fusion proteins comprising a protein or peptidethat protects a plant from a pathogen, the pathogen can be a bacterialpathogen or a fungal pathogen. For example, the pathogen can comprise anα-class Proteobacterium, a β-class Proteobacterium, a γ-classProteobacterium, or a combination thereof. Particular bacterialpathogens include Agrobacterium tumefaciens, Pantoea stewartii, Enviniacarotovora, Ralstonia solanacearum, Pseudomonas syringae, Pseudomonasaeruginosa, Xanthomonas campestris, and combinations thereof.

Other bacterial and fungal pathogens include Acarosporina microspora,Aceria guerreronis, Achlya conspicua, Achlya klebsiana, Achlysiellawilliamsi, Acholeplasmataceae, Acidovorax avenae, Acremonium strictum,Acrocalymma medicaginis, Acrodontium simplex, Acrophialophora fusispora,Acrosporium tingitaninum, Aecidium, Aecidium aechmantherae, Aecidiumamaryllidis, Aecidium breyniae, Aecidium campanulastri, Aecidiumcannabis, Aecidium cantensis, Aecidium caspicum, Aecidium foeniculi,Agrobacterium tumefaciens, Albonectria rigidiuscula, Albugo bliti,Albugo candida, Albugo ipomoeae-panduratae, Albugo laibachii, Albugooccidentalis, Albugo tragopogonis, Alternaria, Alternaria alternata,Alternaria brassicae, Alternaria brassicicola, Alternaria carthami,Alternaria cinerariae, Alternaria citri, Alternaria dauci, Alternariadianthi, Alternaria dianthicola, Alternaria euphorbiicola, Alternariahelianthi, Alternaria helianthicola, Alternaria japonica, Alternarialeucanthemi, Alternaria limicola, Alternaria linicola, Alternaria mali,Alternaria padwickii, Alternaria panax, Alternaria radicina, Alternariaraphani, Alternaria saponariae, Alternaria senecionis, Alternariasolani, Alternaria tenuissima, Alternaria triticina, Alternaria zinniae,Amazonia, Amphobotrys ricini, Anguillosporella vermiformis, Anguina(genus), Anguina agrostis, Anguina amsinckiae, Anguina australis,Anguina balsamophila, Anguina funesta, Anguina graminis, Anguinaspermophaga, Anguina tritici, Anisogramma anomala, Anthostomellapullulans, Antrodia albida, Antrodia serialiformis, Antrodia serialis,Aphanomyces cladogamus, Aphanomyces cochlioides, Aphanomyces euteiches,Aphanomyces euteiches f. sp. pisi, Aphanomyces raphani, Aphelenchoides,Aphelenchoides arachidis, Aphelenchoides besseyi, Aphelenchoidesfragariae, Aphelenchoides parietinus, Aphelenchoides ritzemabosi,Aphelenchus avenae, Apiognomonia errabunda, Apiognomonia veneta,Apiospora montagnei, Appendiculella, Armillaria, Armillaria affinis,Armillaria apalosclera, Armillaria camerunensis, Armillaria duplicate,Armillaria fellea, Armillaria fumosa, Armillaria fuscipes, Armillariagriseomellea, Armillaria heimii, Armillaria mellea, Armillariamelleorubens, Armillaria montagnei, Armillaria omnituens, Armillariapallidula, Armillaria paulensis, Armillaria pelliculata, Armillariaprocera, Armillaria puiggarii, Armillaria singular, Armillaria socialis,Armillaria solidipes, Armillaria tabescens, Armillaria tigrensis,Armillaria umbrinobrunnea, Armillaria viridiflava, Armillaria yungensis,Arthrocladiella, Arthuriomyces peckianus, Ascochyta asparagine,Ascochyta bohemica, Ascochyta caricae, Ascochyta doronici, Ascochytafabae f. sp. lentis, Ascochyta graminea, Ascochyta hordei, Ascochytahumuli, Ascochyta pisi, Ascochyta prasadii, Ascochyta sorghi, Ascochytaspinaciae, Ascochyta tarda, Ascochyta tritici, Ascospora ruborum, Ashbyagossypii, Aspergillus aculeatus, Aspergillus fischerianus, Aspergillusniger, Asperisporium caricae, Asperisporium minutulum, Asteridiella,Asteridiella perseae, Asteroma caryae, Asteroma coryli, Asteromainconspicuum, Athelia arachnoidea, Athelia rolfsii, Aurantiporusfissilis, Belonolaimus, Belonolaimus gracilis, Belonolaimuslongicaudatus, Beniowskia sphaeroidea, Bionectria ochroleuca, Bipolaris,Bipolaris cactivora, Bipolaris cookie, Bipolaris incurvata, Bipolarissacchari, Biscogniauxia capnodes var. capnodes, Biscogniauxia marginata,Biscogniauxia nummularia, Bjerkandera adusta, Blakeslea trispora,Blumeria graminis, Botryodiplodia oncidii, Botryodiplodia ulmicola,Botryosphaeria cocogena, Botryosphaeria corticola, Botryosphaeriadisrupta, Botryosphaeria dothidea, Botryosphaeria marconii,Botryosphaeria obtuse, Botryosphaeria quercuum, Botryosphaeria rhodina,Botryosphaeria ribis, Botryosphaeria stevensii, Botryosporium pulchrum,Botryotinia, Botryotinia fuckeliana, Botrytis anthophila, Botrytiscinerea, Botrytis fabae, Bremia lactucae, Brenneria salicis, Briosiaampelophaga, Bulbomicrosphaera, Burkholderia andropogonis, Burkholderiacaryophylli, Burkholderia glumae, Cadophora malorum, Caespitotheca,Calonectria indusiata, Calonectria kyotensis, Calonectriaquinqueseptata, Calvatia versispora, Camarosporium pistaciae,Camarotella acrocomiae, Camarotella costaricensis, CandidatusLiberibacter, Capitorostrum cocoes, Capnodium footii, Capnodiummangiferum, Capnodium ramosum, Capnodium theae, Caulimoviridae,Cephaleuros virescens, Cephalosporium gramineum, Ceratobasidium cereal,Ceratobasidium cornigerum, Ceratobasidium noxium, Ceratobasidiumramicola, Ceratobasidium setariae, Ceratobasidium stevensii,Ceratocystis adiposa, Ceratocystis coerulescens, Ceratocystis fimbriata,Ceratocystis moniliformis, Ceratocystis paradoxa, Ceratocystis pilifera,Ceratocystis pluriannulata, Ceratorhiza hydrophila, Ceratospermopsis,Cercoseptoria ocellata, Cercospora, Cercospora angreci, Cercospora apii,Cercospora apii f. sp. clerodendri, Cercospora apiicola, Cercosporaarachidicola, Cercospora asparagi, Cercospora atrofiliformis, Cercosporabeticola, Cercospora brachypus, Cercospora brassicicola, Cercosporabrunkii, Cercospora cannabis, Cercospora cantuariensis, Cercosporacapsici, Cercospora carotae, Cercospora corylina, Cercospora fragariae,Cercospora fuchsiae, Cercospora fusca, Cercospora fusimaculans,Cercospora gerberae, Cercospora halstedii, Cercospora handelii,Cercospora hayi, Cercospora hydrangea, Cercospora kikuchii, Cercosporalentis, Cercospora liquidambaris, Cercospora longipes, Cercosporalongissima, Cercospora mamaonis, Cercospora mangiferae, Cercosporamedicaginis, Cercospora melongenae, Cercospora minima, Cercosporaminuta, Cercospora nicotianae, Cercospora odontoglossi, Cercosporapapaya, Cercospora penniseti, Cercospora pisa-sativae, Cercosporaplatanicola, Cercospora puderii, Cercospora pulcherrima, Cercosporarhapidicola, Cercospora rosicola, Cercospora rubrotincta, Cercosporasojina, Cercospora solani, Cercospora solani-tuberosi, Cercosporasorghi, Cercospora theae, Cercospora tuberculans, Cercospora vexans,Cercospora vicosae, Cercospora zeae-maydis, Cercospora zebrina,Cercospora zonata, Cercosporella rubi, Cereal cyst nematode, Ceriporiaspissa, Ceriporia xylostromatoides, Cerrena unicolor, Ceuthospora lauri,Choanephora, Choanephora cucurbitarum, Choanephora infundibulifera,Chondrostereum purpureum, Chrysomyxa ledi var. rhododendri, Chrysomyxaledicola, Chrysomyxa piperiana, Chrysomyxa roanensis, Cladosporium,Cladosporium arthropodii, Cladosporium caryigenum, Cladosporiumcladosporioides, Cladosporium cladosporioides f. sp. pisicola,Cladosporium cucumerinum, Cladosporium herbarum, Cladosporium musae,Cladosporium oncobae, Clavibacter michiganensis, Claviceps fusiformis,Claviceps purpurea, Claviceps sorghi, Claviceps zizaniae, Climacodonpulcherrimus, Climacodon septentrionalis, Clitocybe parasitica,Clonostachys rosea f. rosea, Clypeoporthe iliau, Cochliobolus,Cochliobolus carbonum, Cochliobolus cymbopogonis, Cochliobolushawaiiensis, Cochliobolus heterostrophus, Cochliobolus lunatus,Cochliobolus miyabeanus, Cochliobolus ravenelii, Cochliobolus sativus,Cochliobolus setariae, Cochliobolus spicifer, Cochliobolus stenospilus,Cochliobolus tuberculatus, Cochliobolus victoriae, Coleosporiumhelianthi, Coleosporium ipomoeae, Coleosporium madiae, Coleosporiumpacificum, Coleosporium tussilaginis, Colletotrichum acutatum,Colletotrichum arachidis, Colletotrichum capsici, Colletotrichumcereale, Colletotrichum crassipes, Colletotrichum dematium,Colletotrichum dematium f. spinaciae, Colletotrichum derridis,Colletotrichum destructivum, Colletotrichum fragariae, Colletotrichumgossypii, Colletotrichum higginsianum, Colletotrichum kahawae,Colletotrichum lindemuthianum, Colletotrichum lini, Colletotrichummangenotii, Colletotrichum musae, Colletotrichum nigrum, Colletotrichumorbiculare, Colletotrichum pisi, Colletotrichum sublineolum,Colletotrichum trichellum, Colletotrichum trifolii, Colletotrichumtruncatum, Coniella castaneicola, Coniella diplodiella, Coniellafragariae, Coniothecium chomatosporum, Coniothyrium celtidis-australis,Coniothyrium henriquesii, Coniothyrium rosarum, Coniothyriumwernsdorffiae, Coprinopsis psychromorbida, Cordana johnstonii, Cordanamusae, Coriolopsis floccose, Coriolopsis gallica, Corticium invisum,Corticium penicillatum, Corticium theae, Coryneopsis rubi, Corynesporacassiicola, Coryneum rhododendri, Crinipellis sarmentosa, Cronartiumribicola, Cryphonectriaceae, Cryptocline cyclaminis, Cryptomeliola,Cryptoporus volvatus, Cryptosporella umbrina, Cryptosporiopsistarraconensis, Cryptosporium minimum, Curvularia caricae-papayae,Curvularia penniseti, Curvularia senegalensis, Curvularia trifolii,Cylindrocarpon candidum, Cylindrocarpon ianthothele var. ianthothele,Cylindrocarpon magnusianum, Cylindrocarpon musae, Cylindrocladiellacamelliae, Cylindrocladiella parva, Cylindrocladium clavatum,Cylindrocladium lanceolatum, Cylindrocladium peruvianum, Cylindrocladiumpteridis, Cylindrosporium cannabinum, Cylindrosporium juglandis,Cylindrosporium rubi, Cymadothea trifolii, Cytospora, Cytosporapalmarum, Cytospora personata, Cytospora platani, Cytospora sacchari,Cytospora sacculus, Cytospora terebinthi, Cytosporina ludibunda,Dactuliophora elongata, Daedaleopsis confragosa, Dasineura urticae,Datronia scutellata, Davidiella carinthiaca, Davidiella dianthi,Davidiella tassiana, Deightoniella papuana, Deightoniella torulosa,Dendrophoma marconii, Dendrophora erumpens, Denticularia mangiferae,Dermea pseudotsugae, Diaporthaceae, Diaporthe, Diaporthe arctii,Diaporthe citri, Diaporthe dulcamarae, Diaporthe eres, Diaporthehelianthi, Diaporthe lagunensis, Diaporthe lokoyae, Diaporthe melonis,Diaporthe orthoceras, Diaporthe perniciosa, Diaporthe phaseolorum,Diaporthe phaseolorum var. caulivora, Diaporthe phaseolorum var.phaseolorum, Diaporthe phaseolorum var. sojae, Diaporthe rudis,Diaporthe tanakae, Diaporthe toxica, Dibotryon morbosum, Dicarpelladryina, Didymella bryoniae, Didymella fabae, Didymella lycopersici,Didymosphaeria arachidicola, Didymosphaeria taiwanensis, Dilophosporaalopecuri, Dimeriella sacchari, Diplocarpon earlianum, Diplocarpon mali,Diplocarpon mespili, Diplocarpon rosae, Diplodia laelio-cattleyae,Diplodia manihoti, Diplodia paraphysaria, Diplodia theae-sinensis,Discosia artocreas, Guignardia fulvida, Discostroma corticola,Distocercospora, Distocercospora livistonae, Ditylenchus, Ditylenchusafricanus, Ditylenchus angustus, Ditylenchus destructor, Ditylenchusdipsaci, Dolichodorus heterocephalus, Dothideomycetes, Dothiorellaaromatic, Dothiorella dominicana, Dothiorella gregaria, Dothiorellaulmi, Drechslera avenacea, Drechslera campanulata, Drechsleradematioidea, Drechslera gigantea, Drechslera glycines, Drechsleramusae-sapientium, Drechslera teres f. maculate, Drechslerawirreganensis, Durandiella pseudotsugae, Eballistra lineata, Eballistraoryzae, Eballistraceae, Echinodontium tinctorium, Ectendomeliola,Elsinoe ampelina, Elsinoe australis, Elsinoe batatas, Elsinoebrasiliensis, Elsinoe fawcettii, Elsinoe leucospila, Elsinoe mangiferae,Elsinoe pini, Elsinoe randii, Elsinoe rosarum, Elsinoe sacchari, Elsinoetheae, Elsinoe veneta, Endomeliola, Endothia radicalis, Endothiellagyrosa, Entoleuca mammata, Entorrhizomycetes, Entyloma ageratinae,Entyloma dahlia, Entyloma ellisii, Epicoccum nigrum, Ergot, Erwinia,Envinia chrysanthemi, Erwinia psidii, Erysiphaceae, Erysiphales,Erysiphe, Erysiphe alphitoides, Erysiphe betae, Erysiphebrunneopunctata, Erysiphe cichoracearum, Erysiphe cruciferarum, Erysipheflexuosa, Erysiphe graminis f. sp. avenae, Erysiphe graminis f. sp.tritici, Erysiphe heraclei, Erysiphe pisi, Eutypella parasitica,Eutypella scoparia, Exobasidium burtii, Exobasidium reticulatum,Exobasidium vaccinii var. japonicum, Exobasidium vaccinii-uliginosi,Exobasidium vexans, Exophiala, Flavescence doree, Fomes fasciatus, Fomeslamaensis, Fomes meliae, Fomitopsis cajanderi, Fomitopsis palustris,Fomitopsis rosea, Fomitopsis spraguei, Fomitopsis supina, Formaspecialis, Frommeella tormentillae, Fusarium, Fusarium affine, Fusariumarthrosporioides, Fusarium circinatum, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium incarnatum, Fusarium solani,Fusarium merismoides, Fusarium oxysporum f. sp. albedinis, Fusariumoxysporum f. sp. asparagi, Fusarium oxysporum f. sp. batatas, Fusariumoxysporum f. sp. betae, Fusarium oxysporum f. sp. cannabis, Fusariumoxysporum f. sp. citri, Fusarium oxysporum f. sp. coffea, Fusariumoxysporum f. sp. cubense, Fusarium oxysporum f. sp. cyclaminis, Fusariumoxysporum f. sp. dianthi, Fusarium oxysporum f. sp. lentis, Fusariumoxysporum f. sp. lini, Fusarium oxysporum f. sp. lycopersici, Fusariumoxysporum f. sp. medicaginis, Fusarium oxysporum f. sp. pisi, Fusariumoxysporum f. sp. radicis-lycopersici, Fusarium pallidoroseum, Fusariumproliferatum, Fusarium redolens, Fusarium sacchari, Fusarium solani f.sp. pisi, Fusarium sporotrichioides, Fusarium subglutinans, Fusariumsulphureum, Fuscoporia torulosa, Fusicladium pisicola, Fusicoccumaesculi, Fusicoccum amygdali, Gaeumannomyces graminis var tritici,Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var.graminis, Galactomyces candidum, Ganoderma brownii, Ganoderma lobatum,Ganoderma orbiforme, Ganoderma philippii, Ganoderma tornatum, Ganodermazonatum, Geastrumia polystigmatis, Georgefischeriaceae,Georgefischeriales, Geosmithia morbida, Geotrichum, Geotrichum candidum,Geotrichum candidum var. citri-aurantii, Geotrichum klebahnii,Gibberella, Gibberella acuminata, Gibberella avenacea, Gibberellabaccata, Gibberella cyanogena, Gibberella fujikuroi, Gibberellafujikuroi var. subglutinans, Gibberella intricans, Gibberella pulicaris,Gibberella stilboides, Gibberella xylarioides, Gibberella zeae,Gibellina cerealis, Gilbertella persicaria, Gjaerumiaceae, Gliocladiumvermoeseni, Globodera pallida, Globodera rostochiensis, Globoderatabacum, Gloeocercospora sorghi, Gloeocystidiellum porosum, Gloeophyllummexicanum, Gloeophyllum trabeum, Gloeoporus dichrous, Gloeosporiumcattleyae, Gloeosporium theae-sinensis, Glomerella cingulate, Glomerellaglycines, Glomerella graminicola, Glomerella tucumanensis, Gnomoniacaryae, Gnomonia comari, Gnomonia dispora, Gnomonia iliau, Gnomonialeptostyla, Gnomonia nerviseda, Gnomonia rubi, Golovinomycescichoracearum var. latisporus, Granulobasidium vellereum, Graphiolaphoenicis, Graphium rigidum, Graphium rubrum, Graphyllium pentamerum,Grovesinia pyramidalis, Guignardia bidwellii f. muscadinii, Guignardiacamelliae, Guignardia citricarpa, Guignardia mangiferae, Guignardiamusae, Guignardia philoprina, Gummosis, Gymnoconia nitens, Gymnopusdryophilus, Gymnosporangium clavipes, Gymnosporangium sabinae,Gymnosporangium globosum, Gymnosporangium jumperi-virginianae,Gymnosporangium kernianum, Gymnosporangium nelsonii, Gymnosporangiumyamadae, Haematonectria haematococca, Hansenula subpelliculosa,Hapalosphaeria deformans, Haplobasidion musae, Haustorium,Helicobasidium compactum, Helicobasidium longisporum, Helicobasidiumpurpureum, Helicoma muelleri, Helicotylenchus, Helicotylenchusdihystera, Helicotylenchus multicinctus, Helminthosporium cookei,Helminthosporium papulosum, Helminthosporium solani, Helotiales,Hemicriconemoides kanayaensis, Hemicriconemoides mangiferae,Hemicycliophora arenaria, Hemlock woolly adelgid, Hendersoniacreberrima, Hendersonia theicola, Hericium coralloides, Heterobasidionannosum, Heterodera, Heterodera amygdali, Heterodera arenaria,Heterodera aucklandica, Heterodera avenae, Heterodera bergeniae,Heterodera bifenestra, Heterodera cacti, Heterodera cajani, Heteroderacanadensis, Heterodera cardiolata, Heterodera carotae, Heteroderaciceri, Heterodera cruciferae, Heterodera delvii, Heterodera elachista,Heterodera filipjevi, Heterodera gambiensis, Heterodera goettingiana,Heterodera hordecalis, Heterodera humuli, Heterodera latipons,Heterodera medicaginis, Heterodera oryzae, Heterodera oryzicola,Heterodera rosii, Heterodera sacchari, Heterodera schachtii, Heteroderatabacum, Heterodera trifolii, Heteroderidae, Hexagonia hydnoides,Hirschmanniella oryzae, Hoplalaimus galeatus, Hoplolaimidae, Hoplolaimuscolumbus, Hoplolaimus indicus, Hoplolaimus magnistylus, Hoplolaimuspararobustus, Hoplolaimus seinhorsti, Hoplolaimus uniformis,Huanglongbing, Hyaloperonospora, Hyaloperonospora arabidopsidis,Hyaloperonospora brassicae, Hyaloperonospora parasitica, Hymenulaaffinis, Hyphodermella corrugata, Hyphodontia aspera, Hyphodontiasambuci, Hypochnus, Hypoxylon tinctor, Idriella lunata, Inonotusarizonicus, Inonotus cuticularis, Inonotus dryophilus, Inonotushispidus, Inonotus ludovicianus, Inonotus munzii, Inonotus tamaricis,Irenopsis, Irpex destruens, Irpex lacteus, Isariopsis clavispora,Johncouchia mangiferae, Kabatiella caulivora, Kabatiella lini, Karnalbunt, Khuskia oryzae, Kretzschmaria deusta, Kretzschmaria zonata,Kuehneola uredinis, Kutilakesa pironii, Labrella coryli, Laeticorticiumroseum, Laetiporus baudonii, Lagenocystis radicicola, Laricifomesofficinalis, Lasiodiplodia theobromas, Leandria momordicae, Leifsoniaxyli xyli, Lentinus tigrinus, Lenzites betulina, Lenzites elegans,Lepteutypa cupressi, Leptodontidium elatius var. elatius, Leptographiummicrosporum, Leptosphaeria acuta, Leptosphaeria cannabina, Leptosphaeriaconiothyrium, Leptosphaeria libanotis, Leptosphaeria lindquistii,Leptosphaeria maculans, Leptosphaeria musarum, Leptosphaeria pratensis,Leptosphaeria sacchari, Leptosphaeria woroninii, Leptosphaerulinacrassiasca, Leptosphaerulina trifolii, Leptothyrium nervisedum,Leptotrochila medicaginis, Leucocytospora leucostoma, Leucostomaauerswaldii, Leucostoma kunzei, Leucostoma persoonii, Leveillulacompositarum f. helianthi, Leveillula leguminosarum f. lentis,Leveillula taurica, Ligniera pilorum, Limacinula tenuis, Linochoragraminis, Longidorus africanus, Longidorus maximus, Longidorus sylphus,Lopharia crassa, Lophodermium, Lophodermium aucupariae, Lophodermiumschweinitzii, Lophodermium seditiosum, Macrophoma mangiferae, Macrophomatheicola, Macrophomina phaseolina, Macrosporium cocos, Magnaporthe,Magnaporthe grisea, Magnaporthe salvinii, Mamianiella coryli,Marasmiellus cocophilus, Marasmiellus inoderma, Marasmiellus scandens,Marasmiellus stenophyllus, Marasmius crinis-equi, Marasmius sacchari,Marasmius semiustus, Marasmius stenophyllus, Marasmius tenuissimus,Massarina walkeri, Mauginiella scaettae, Melampsora, Melampsora linivar. lini, Melampsora medusae, Melampsora occidentalis, Melanconiscarthusiana, Melanconium juglandinum, Meliola, Meliola mangiferae,Meliolaceae, Meloidogyne acronea, Meloidogyne arenaria, Meloidogyneartiellia, Meloidogyne brevicauda, Meloidogyne chitwoodi, Meloidogyneenterolobii, Meloidogyne fruglia, Meloidogyne gajuscus, Meloidogyneincognita, Meloidogyne javanica, Meloidogyne naasi, Meloidogynepartityla, Meloidogyne thamesi, Meripilus giganteus, Merliniusbrevidens, Meruliopsis ambigua, Mesocriconema xenoplax, Microascusbrevicaulis, Microbotryum violaceum, Microdochium bolleyi, Microdochiumdimerum, Microdochium panattonianum, Microdochium phragmitis,Microsphaera, Microsphaera coryli, Microsphaera diffusa, Microsphaeraellisii, Microsphaera euphorbiae, Microsphaera hommae, Microsphaerapenicillata, Microsphaera penicillata var. vaccinii, Microsphaeravaccinii, Microsphaera verruculosa, Microstroma juglandis, Moesziomycesbullatus, Monilinia azaleae, Monilinia fructicola, Monilinia fructigena,Monilinia laxa, Monilinia mali, Moniliophthora perniciosa,Moniliophthora roreri, Monilochaetes infuscans, Monochaetia coryli,Monochaetia mali, Monographella albescens, Monographella cucumerina,Monographella nivalis var. neglecta, Monographella nivalis var. nivalis,Mononegavirales, Monosporascus cannonballus, Monosporascus eutypoides,Monostichella coryli, Mucor circinelloides, Mucor hiemalis, Mucorhiemalis f. silvaticus, Mucor mucedo, Mucor paronychius, Mucorpiriformis, Mucor racemosus, Mycena citricolor, Mycena maculate,Mycocentrospora acerina, Mycoleptodiscus terrestris, Mycosphaerellaangulata, Mycosphaerella arachidis, Mycosphaerella areola,Mycosphaerella berkeleyi, Mycosphaerella bolleana, Mycosphaerellabrassicicola, Mycosphaerella caricae, Mycosphaerella caryigena,Mycosphaerella cerasella, Mycosphaerella citri, Mycosphaerellacoffeicola, Mycosphaerella confusa, Mycosphaerella cruenta,Mycosphaerella dendroides, Mycosphaerella eumusae, Mycosphaerellafragariae, Mycosphaerella gossypina, Mycosphaerella graminicola,Mycosphaerella henningsii, Mycosphaerella horii, Mycosphaerellajuglandis, Mycosphaerella lageniformis, Mycosphaerella linicola,Mycosphaerella louisianae, Mycosphaerella musae, Mycosphaerellamusicola, Mycosphaerella palmicola, Mycosphaerella pinodes,Mycosphaerella pistaciarum, Mycosphaerella pistacina, Mycosphaerellaplatanifolia, Mycosphaerella polymorpha, Mycosphaerella pomi,Mycosphaerella punctiformis, Mycosphaerella pyri, Didymella rabiei,Mycosphaerella recutita, Mycosphaerella rosicola, Mycosphaerella rubi,Mycosphaerella stigmina-platani, Mycosphaerella striatiformans,Mycovellosiella concors, Mycovellosiella fulva, Mycovellosiella koepkei,Mycovellosiella vaginae, Myriogenospora aciculispora, Myrotheciumroridum, Myrothecium verrucaria, Nacobbus aberrans, Nacobbus dorsalis,Naevala perexigua, Naohidemyces vaccinii, Nectria, Nectria cinnabarina,Nectria coccinea, Nectria ditissima, ectria foliicola, Nectria mammoideavar. rubi, Nectria mauritiicola, Nectria peziza, Nectria pseudotrichia,Nectria radicicola, Nectria ramulariae, Nectriella pironii, Nemaniadiffusa, Nemania serpens var. serpens, Nematospora coryli, Neocosmosporavasinfecta, Neodeightonia phoenicum, Neoerysiphe, Neofabraeamalicorticis, Neofabraea perennans, Neofusicoccum mangiferae, Neonectriagalligena, Oidiopsis gossypii, Oidium (genus), Oidium arachidis, Oidiumcaricae-papayae, Oidium indicum, Oidium mangiferae, Oidium manihotis,Oidium tingitaninum, Olpidium brassicae, Omphalia tralucida,Oncobasidium theobromae, Onnia tomentosa, Ophiobolus anguillides,Ophiobolus cannabinus, Ophioirenina, Ophiostoma ulmi, Ophiostomawageneri, Ovulariopsis papayae, Ovulinia azaleae, Ovulitis azaleae,Oxyporus corticola, Oxyporus latemarginatus, Oxyporus populinus,Oxyporus similis, Ozonium texanum var. parasiticum, Paecilomyces fulvus,Paralongidorus maximus, Paratrichodorus christiei, Paratrichodorusminor, Paratylenchus curvitatus, Paratylenchus elachistus, Paratylenchushamatus, Paratylenchus macrophallus, Paratylenchus microdorus,Paratylenchus projectus, Paratylenchus tenuicaudatus, Pathovar, Pauahia,Peach latent mosaic viroid, Pectobacterium carotovorum, Peltasterfructicola, Penicillium aurantiogriseum, Penicillium digitatum,Penicillium expansum, Penicillium funiculosum, Penicillium glabrum,Penicillium italicum, Penicillium purpurogenum, Penicillium ulaiense,Peniophora, Peniophora albobadia, Peniophora cinerea, Peniophoraquercina, Peniophora sacrata, Perenniporia fraxinea, Perenniporiafraxinophila, Perenniporia medulla-panis, Perenniporia subacida,Periconia circinata, Periconiella cocoes, Peridermium californicum,Peronosclerospora miscanthi, Peronosclerospora sacchari,Peronosclerospora sorghi, Peronospora, Peronospora anemones, Peronosporaantirrhini, Peronospora arborescens, Peronospora conglomerata,Peronospora destructor, Peronospora dianthi, Peronospora dianthicola,Peronospora farinosa, Peronospora farinosa f. sp. betae, Peronosporahyoscyami f. sp. tabacina, Peronospora manshurica, Peronosporapotentillae, Peronospora sparsa, Peronospora trifoliorum, Peronosporavalerianellae, Peronospora viciae, Pestalosphaeria concentrica,Pestalotia longiseta, Pestalotia longisetula, Pestalotia rhododendri,Pestalotiopsis, Pestalotiopsis adusta, Pestalotiopsis arachidis,Pestalotiopsis disseminata, Pestalotiopsis guepini, Pestalotiopsisleprogena, Pestalotiopsis longiseta, Pestalotiopsis mangiferae,Pestalotiopsis palmarum, Pestalotiopsis sydowiana, Pestalotiopsis theae,Pestalotiopsis versicolor, Phacidiopycnis padwickii, Phacidiuminfestans, Phaeochoropsis mucosa, Phaeocytostroma iliau, Phaeocytostromasacchari, Phaeoisariopsis bataticola, Phaeolus schweinitzii,Phaeoramularia angolensis, Phaeoramularia dissiliens, Phaeoramulariaheterospora, Phaeoramularia manihotis, Phaeoseptoria musae,Phaeosphaerella mangiferae, Phaeosphaerella theae, Phaeosphaeriaavenaria f. sp. avenaria, Phaeosphaeria avenaria f. sp. triticae,Phaeosphaeria herpotrichoides, Phaeosphaeria microscopica, Phaeosphaerianodorum, Phaeosphaeriopsis obtusispora, Phaeotrichoconis crotalariae,Phakopsora gossypii, Phakopsora pachyrhizi, Phanerochaete allantospora,Phanerochaete arizonica, Phanerochaete avellanea, Phanerochaete burtii,Phanerochaete carnosa, Phanerochaete chrysorhizon, Phanerochaeteradicata, Phanerochaete salmonicolor, Phanerochaete tuberculata,Phanerochaete velutina, Phellinus ferreus, Phellinus gilvus, Phellinusigniarius, Phellinus pini, Phellinus pomaceus, Phellinus weirii,Phialophora asteris, Phialophora cinerescens, Phialophora gregata,Phialophora tracheiphila, Phloeospora multimaculans, Pholiotavariicystis, Phoma, Phoma caricae-papayae, Phoma clematidina, Phomacostaricensis, Phoma cucurbitacearum, Phoma destructiva, Phoma draconis,Phoma eupyrena, Phoma exigua, Phoma exigua var. exigua, Phoma exiguavar. foveata, Phoma exigua var. linicola, Phoma glomerata, Phomaglycinicola, Phoma herbarum, Phoma insidiosa, Phoma medicaginis, Phomamicrospora, Phoma nebulosa, Phoma oncidii-sphacelati, Phoma pinodella,Phoma scabra, Phoma sclerotioides, Phoma strasseri, Phoma tracheiphila,Phomopsis arnoldiae, Phomopsis asparagi, Phomopsis asparagicola,Phomopsis azadirachtae, Phomopsis cannabina, Phomopsis caricae-papayae,Phomopsis coffeae, Phomopsis elaeagni, Phomopsis ganjae, Phomopsisjavanica, Phomopsis lokoyae, Phomopsis mangiferae, Phomopsis obscurans,Phomopsis perseae, Phomopsis prunorum, Phomopsis scabra, Phomopsissclerotioides, Phomopsis tanakae, Phomopsis theae, Photoassimilate,Phragmidium, Phragmidium mucronatum, Phragmidium rosae-pimpinellifoliae,Phragmidium rubi-idaei, Phragmidium violaceum, Phyllachora cannabis,Phyllachora graminis var. graminis, Phyllachora gratissima, Phyllachoramusicola, Phyllachora pomigena, Phyllachora sacchari, Phyllactinia,Phyllactinia angulata, Phyllactinia guttata, Phyllody, Phyllosticta,Phyllosticta alliariaefoliae, Phyllosticta anacardiacearum, Phyllostictaarachidis-hypogaeae, Phyllosticta batatas, Phyllosticta capitalensis,Phyllosticta caricae-papayae, Phyllosticta carpogena, Phyllostictacircumscissa, Phyllosticta coffeicola, Phyllosticta concentrica,Phyllosticta coryli, Phyllosticta cucurbitacearum, Phyllostictacyclaminella, Phyllosticta erratica, Phyllosticta hawaiiensis,Phyllosticta lentisci, Phyllosticta manihotis, Phyllosticta micropuncta,Phyllosticta mortonii, Phyllosticta nicotianae, Phyllosticta palmetto,Phyllosticta penicillariae, Phyllosticta perseae, Phyllosticta platani,Phyllosticta pseudocapsici, Phyllosticta sojaecola, Phyllostictasolitaria, Phyllosticta theae, Phyllosticta theicola, Phymatotrichopsisomnivora, Physalospora abdita, Physalospora disrupta, Physalosporaperseae, Physarum cinereum, Physoderma alfalfae, Physoderma leproides,Physoderma trifolii, Physopella ampelopsidis, Phytophthora, Phytophthoraalni, Phytophthora boehmeriae, Phytophthora cactorum, Phytophthoracajani, Phytophthora cambivora, Phytophthora capsici, Phytophthoracinnamomi, Phytophthora citricola, Phytophthora citrophthora,Phytophthora cryptogea, Phytophthora drechsleri, Phytophthoraerythroseptica, Phytophthora fragariae, Phytophthora fragariae var.rubi, Phytophthora gallica, Phytophthora hibernalis, Phytophthorainfestans, Phytophthora inflata, Phytophthora iranica, Phytophthorakatsurae, Phytophthora kernoviae, Phytophthora lateralis, Phytophthoramedicaginis, Phytophthora megakarya, Phytophthora megasperma,Phytophthora nicotianae, Phytophthora palmivora, Phytophthora phaseoli,Phytophthora plurivora, Phytophthora ramorum, Phytophthora sojae,Phytophthora syringae, Phytophthora tentaculata, Phytoplasma, Pichiamembranifaciens, Pichia subpelliculosa, Pileolaria terebinthi,Pilidiella quercicola, Plasmodiophora brassicae, Plasmopara, Plasmoparahalstedii, Plasmopara helianthi f. helianthi, Plasmoparalactucae-radicis, Plasmopara nivea, Plasmopara obducens, Plasmoparapenniseti, Plasmopara pygmaea, Plasmopara viticola, Platychora ulmi,Plenodomus destruens, Plenodomus meliloti, Pleochaeta, Pleosphaerulinasojicola, Pleospora alfalfae, Pleospora betae, Pleospora herbarum,Pleospora lycopersici, Pleospora tarda, Pleospora theae, Pleurotusdryinus, Podosphaera, Podosphaera clandestina var. clandestine,Podosphaera fusca, Podosphaera leucotricha, Podosphaera macularis,Podosphaera pannosa, Podosphaera tridactyla, Podosphaera tridactyla var.tridactyla, Podosphaera xanthii, Polymyxa graminis, Polyscytalumpustulans, Polystigma fulvum, Poria hypobrunnea, Postia tephroleuca,Potato cyst nematode, Pratylenchus alleni, Pratylenchus brachyurus,Pratylenchus coffeae, Pratylenchus crenatus, Pratylenchus dulscus,Pratylenchus fallax, Pratylenchus flakkensis, Pratylenchus goodeyi,Pratylenchus hexincisus, Pratylenchus loosi, Pratylenchus minutus,Pratylenchus mulchandi, Pratylenchus musicola, Pratylenchus neglectus,Pratylenchus penetrans, Pratylenchus pratensis, Pratylenchus reniformia,Pratylenchus scribneri, Pratylenchus thornei, Pratylenchus vulnus,Pratylenchus zeae, Pseudocercospora, Pseudocercospora arecacearum,Pseudocercospora cannabina, Pseudocercospora fuligena, Pseudocercosporagunnerae, Pseudocercospora kaki, Pseudocercospora mali, Pseudocercosporapandoreae, Pseudocercospora puderi, Pseudocercospora purpurea,Pseudocercospora rhapisicola, Pseudocercospora subsessilis,Pseudocercospora theae, Pseudocercospora vitis, Pseudocercosporellacapsellae, Pseudocochliobolus eragrostidis, Pseudoepicoccum cocos,Pseudomonas amygdali, Pseudomonas asplenii, Pseudomonas avellanae,Pseudomonas caricapapayae, Pseudomonas cichorii, Pseudomonascoronafaciens, Pseudomonas corrugate, Pseudomonas ficuserectae,Pseudomonas flavescens, Pseudomonas fuscovaginae, Pseudomonas helianthi,Pseudomonas marginalis, Pseudomonas meliae, Pseudomonas oryzihabitans,Pseudomonas palleroniana, Pseudomonas papaveris, Pseudomonas salomonii,Pseudomonas savastanoi, Pseudomonas syringae, Pseudomonas tomato,Pseudomonas tremae, Pseudomonas turbinellae, Pseudomonas viridiflava,Pseudoperonospora cannabina, Pseudoperonospora cubensis,Pseudoperonospora humuli, Pseudopezicula tetraspora, Pseudopeziculatracheiphila, Pseudopeziza jonesii, Pseudopeziza medicaginis,Pseudopeziza trifolii, Pseudoseptoria donacis, Puccinia, Pucciniaangustata, Puccinia arachidis, Puccinia aristidae, Puccinia asparagi,Puccinia cacabata, Puccinia campanulae, Puccinia carthami, Pucciniacoronate, Puccinia coronata var. hordei, Puccinia dioicae, Pucciniaerianthi, Puccinia extensicola var. hieraciata, Puccinia helianthi,Puccinia hordei, Puccinia jaceae var. solstitialis, Puccinia kuehnii,Puccinia mariae-wilsoniae, Puccinia melanocephala, Puccinia menthae,Puccinia pelargonii-zonalis, Puccinia pittieriana, Puccinia poarum,Puccinia psidii, Puccinia purpurea, Puccinia recondita, Pucciniaschedonnardii, Puccinia sessilis, Puccinia striiformis f. sp. hordei,Puccinia striiformis var. striiformis, Puccinia subnitens, Pucciniasubstriata var. indica, Puccinia verruca, Puccinia xanthii,Pucciniaceae, Pucciniastrum, Pucciniastrum americanum, Pucciniastrumarcticum, Pucciniastrum coryli, Pucciniastrum epilobii, Pucciniastrumhydrangeas, Punctodera chalcoensis, Pycnoporus cinnabarinus, Pycnoporussanguineus, Pycnostysanus azaleae, Pyrenochaeta lycopersici,Pyrenochaeta terrestris, Pyrenopeziza brassicae, Pyrenophora,Pyrenophora avenae, Pyrenophora chaetomioides, Pyrenophora graminea,Pyrenophora seminiperda, Pyrenophora teres, Pyrenophora teres f.maculata, Pyrenophora teres f. teres, Pyrenophora tritici-repentis,Pythiaceae, Pythiales, Pythium, Pythium acanthicum, Pythiumaphanidermatum, Pythium aristosporum, Pythium arrhenomanes, Pythiumbuismaniae, Pythium debaryanum, Pythium deliense, Pythium dissotocum,Pythium graminicola, Pythium heterothallicum, Pythium hypogynum, Pythiumirregulare, Pythium iwayamae, Pythium mastophorum, Pythium middletonii,Pythium myriotylum, Pythium okanoganense, Pythium paddicum, Pythiumparoecandrum, Pythium perniciosum, Pythium rostratum, Pythiumscleroteichum, Pythium spinosum, Pythium splendens, Pythium sulcatum,Pythium sylvaticum, Pythium tardicrescens, Pythium tracheiphilum,Pythium ultimum, Pythium ultimum var. ultimum, Pythium vexans, Pythiumviolae, Pythium volutum, Quinisulcius acutus, Quinisulcius capitatus,Radopholous similis, Radopholus similis, Ralstonia solanacearum,Ramichloridium musae, Ramularia, Ramularia beticola, Ramularia brunnea,Ramularia coryli, Ramularia cyclaminicola, Ramularia macrospora,Ramularia menthicola, Ramularia necator, Ramularia primulae, Ramulariaspinaciae, Ramularia subtilis, Ramularia tenella, Ramulispora sorghi,Ramulispora sorghicola, Resinicium bicolor, Rhabdocline pseudotsugae,Rhabdocline weirii Rhabdoviridae, Rhinocladium corticola, Rhizoctonia,Rhizoctonia leguminicola, Rhizoctonia rubi, Rhizoctonia solani,Rhizomorpha subcorticalis, Rhizophydium graminis, Rhizopus arrhizus,Rhizopus circinans, Rhizopus microsporus var. macrospores, Rhizopusoryzae, Rhodococcus fascians, Rhynchosporium, Rhynchosporium secalis,Rhytidhysteron rufulum, Rhytisma acerinum, Rhytisma vitas, Rigidoporuslineatus, Rigidoporus microporus, Rigidoporus ulmarius, Rigidoporusvinctus, Rosellinia arcuata, Rosellinia bunodes, Rosellinia necatrix,Rosellinia pepo, Rosellinia subiculata, Rotylenchulus, Rotylenchulusparvus, Rotylenchulus reniformis, Rotylenchus brachyurus, Rotylenchusrobustus, Saccharicola taiwanensis, Saccharomyces florentinus,Saccharomyces kluyveri, Sarocladium oryzae, Saw adaea, Sawadaeatulasnei, Schiffnerula cannabis, Schizoparme straminea, Schizophyllumcommune, Schizopora flavipora, Schizothyrium pomi, Scleroderris canker,Sclerophthora macrospora, Sclerophthora rayssiae, Sclerosporagraminicola, Sclerospora mischanthi, Sclerotinia borealis, Sclerotiniaminor, Sclerotinia ricini, Sclerotinia sclerotiorum, Sclerotiniaspermophila, Sclerotinia trifoliorum, Sclerotium, Sclerotium cinnamomi,Sclerotium delphinii, Scutellonema brachyurum, Scutellonema cavenessi,Scytinostroma galactinum, Seimatosporium mariae, Seimatosporiumrhododendri, Selenophoma linicola, Septobasidium, Septobasidiumbogoriense, Septobasidium pilosum, Septobasidium pseudopedicellatum,Septobasidium theae, Septocyta ruborum, Septogloeum potentillae,Septoria, Septoria aciculosa, Septoria ampelina, Septoria azalea,Septoria bataticola, Septoria campanulae, Septoria cannabis, Septoriacaryae, Septoria citri, Septoria cucurbitacearum, Septoria darrowii,Septoria dianthi, Septoria eumusae, Septoria fragariae, Septoriafragariaecola, Septoria glycines, Septoria helianthi, Septoria humuli,Septoria hydrangeas, Septoria lactucae, Septoria liquidambaris, Septorialycopersici, Septoria lycopersici var. malagutii, Septoria menthae,Septoria ostryae, Septoria passerinii, Septoria pisi, Septoriapistaciae, Septoria platanifolia, Septoria rhododendri, Septoriasecalis, Septoria selenophomoides, Setosphaeria rostrata, Setosphaeriaturcica, Sirosporium diffusum, Sparassis, Sphaceloma, Sphacelomaarachidis, Sphaceloma coryli, Sphaceloma menthae, Sphaceloma perseae,Sphaceloma poinsettiae, Sphaceloma pyrinum, Sphaceloma randii,Sphaceloma sacchari, Sphaceloma theae, Sphacelotheca reiliana,Sphaerella platanifolia, Sphaeropsis tumefaciens, Sphaerotheca,Sphaerotheca castagnei, Sphaerotheca fitliginea, Sphaerulina oryzina,Sphaerulina rehmiana, Sphaerulina rubi, Sphenospora kevorkianii,Spiniger meineckellus, Spiroplasma, Spongipellis unicolor, Sporisoriumcruentum, Sporisorium ehrenbergi, Sporisorium scitamineum, Sporisoriumsorghi, Sporonema phacidioides, Stagonospora avenae f. sp. triticae,Stagonospora meliloti, Stagonospora recedens, Stagonospora sacchari,Stagonospora tainanensis, Steccherinum ochraceum, Stegocintractia junci,Stegophora ulmea, Stemphylium alfalfa, Stemphylium bolickii, Stemphyliumcannabinum, Stemphylium globuliferum, Stemphylium lycopersici,Stemphylium sarciniforme, Stemphylium solani, Stemphylium vesicarium,Stenella anthuriicola, Stereum, Stereum hirsutum, Stereum rameale,Stereum sanguinolentum, Stigmatomycosis, Stigmella platani-racemosae,Stigmina carpophila, Stigmina liquidambaris, Stigmina palmivora,Stigmina platani, Stigmina platani-racemosae, Subanguina radicicola,Subanguina wevelli, Sydowia polyspora, Sydowiella depressula,Sydowiellaceae, Synchytrium endobioticum, Synchytrium fragariae,Synchytrium liquidambaris, Taiwanofungus camphoratus, Tapesia acuformis,Tapesia yallundae, Taphrina aurea, Taphrina bullata, Taphrinacaerulescens, Taphrina coryli, Taphrina deformans, Taphrina entomospora,Taphrina johansonii, Taphrina potentillae, Taphrina ulmi, Taphrinawiesneri, Thanatephorus cucumeris, Thielaviopsis, Thielaviopsisbasicola, Thyrostroma compactum, Tilletia barclayana, Tilletia caries,Tilletia controversa, Tilletia laevis, Tilletia tritici, Tilletiawalkeri, Tilletiariaceae, Tobacco necrosis virus, Togniniaceae,Trachysphaera fructigena, Trametes gibbosa, Trametes hirsute, Trametesnivosa, Trametes pubescens, Tranzschelia discolor fsp. persica,Tranzschelia pruni-spinosae var. discolor, Trichaptum biforme,Trichoderma harzianum, Trichoderma koningii, Trichoderma viride,Trichothecium roseum, Tripospermum acerinum, Truncatella, Truncatellalaurocerasi, Tubercularia lateritia, Tubercularia ulmea, Tubeufiapezizula, Tunstallia aculeata, Tylenchorhynchus, Tylenchorhynchusbrevilineatus, Tylenchorhynchus claytoni, Tylenchorhynchus dubius,Tylenchorhynchus maximus, Tylenchorhynchus nudus, Tylenchorhynchusphaseoli, Tylenchorhynchus vulgaris, Tylenchorhynchus zeae, Tylenchulussemipenetrans, Typhula idahoensis, Typhula incarnate, Typhulaishikariensis, Typhula ishikariensis var. canadensis, Typhulavariabilis, Typhulochaeta, Tyromyces calkinsii, Tyromyces chioneus,Tyromyces galactinus, Ulocladium atrum, Ulocladium consortiale,Uncinula, Uncinula macrospora, Uncinula necator, Uredo behnickiana,Uredo kriegeriana, Uredo musae, Uredo nigropuncta, Uredo rangelii,Urocystis, Urocystis agropyri, Urocystis brassicae, Urocystis occulta,Uromyces, Uromyces apiosporus, Uromyces beticola, Uromycesciceris-arietini, Uromyces dianthi, Uromyces euphorbiae, Uromycesgraminis, Uromyces inconspicuus, Uromyces lineolatus subsp. nearcticus,Uromyces medicaginis, Uromyces musae, Uromyces oblongus, Uromycespisi-sativi, Uromyces proëminens var. poinsettiae, Uromycestrifolii-repentis var. fallens, Uromyces viciae-fabae var. viciae-fabae,Urophlyctis leproides, Urophlyctis trifolii, Urophora cardui,Ustilaginales, Ustilaginoidea virens, Ustilaginomycetes, Ustilago,Ustilago avenae, Ustilago hordei, Ustilago maydis, Ustilago nigra,Ustilago nuda, Ustilago scitaminea, Ustilago tritici, Valsa abietis,Valsa ambiens, Valsa auerswaldii, Valsa ceratosperma, Valsa kunzei,Valsa nivea, Valsa sordida, Valsaria insitiva, Venturia carpophila,Venturia inaequalis, Venturia pirina, Venturia pyrina, Veronaea musae,Verticillium, Verticillium albo-atrum, Verticillium albo-atrum var.menthae, Verticillium dahliae, Verticillium longisporum, Verticilliumtheobromas, Villosiclava virens, Virescence, Waitea circinata,Wuestneiopsis Georgiana, Xanthomonas ampelina, Xanthomonas axonopodis,Xanthomonas campestris, Xanthomonas campestris pv. campestris,Xanthomonas oryzae, Xeromphalina fraxinophila, Xiphinema americanum,Xiphinema bakeri, Xiphinema brevicolle, Xiphinema diversicaudatum,Xiphinema insigne, Xiphinema rivesi, Xiphinema vuittenezi, Xylaria mali,Xylaria polymorpha, Xylella fastidiosa, Xylophilus, Xylophilusampelinus, Zopfia rhizophila, Zygosaccharomyces bailii, andZygosaccharomyces florentinus.

Insect and worm pathogens include Acalymma, Acyrthosiphon pisum, Africanarmyworm, Africanized bee, Agromyzidae, Agrotis munda, Agrotisporphyricollis, Aleurocanthus woglumi, Aleyrodes proletella, Alphitobiusdiaperinus, Altica chalybea, Anasa tristis, Anguina tritici, Anisopliaaustriaca, Anthonomus pomorum, Anthonomus signatus, Aonidiella aurantii,Apamea apamiformis, Apamea niveivenosa, Aphelenchoides spp., aphid,Aphis gossypii, apple maggot, Argentine ant, army cutworm, Arotrophoraarcuatalis, Asterolecanium coffeae, Athous haemorrhoidalis, Aulacophora,Australian plague locust, Bactericera cockerelli, Bactrocera, Bactroceracorrecta, Bagrada hilaris, banded hickory borer, beet armyworm,Belonolaimus spp., black bean aphid, Blepharidopterus chlorionis, Bogongmoth, boll weevil, Bradysia similigibbosa, Brassica pod midge,Brevicoryne brassicae, brown locust, brown marmorated stink bug, brownplanthopper, Bursephelenchus spp., cabbage Moth, cabbage worm,Callosobruchus maculatus, cane beetle, carrot fly, cereal cystnematodes, Cecidomyiidae, Ceratitis capitata, Ceratitis rosa, cerealleaf beetle, Chlorops pumilionis, citrus long-horned beetle, Coccusviridis, codling moth, coffee borer beetle, colorado potato beetle,confused flour beetle, crambus, cucumber beetle, Curculio nucum,Curculio occidentis, cutworm, Cyclocephala borealis, dark sword-grass,date stone beetle, Delia spp., Delia antiqua, Delia floralis, Deliaradicum, Desert locust, Diabrotica, Diabrotica balteata, Diabroticaspeciosa, diamondback moth, Diaphania indica, Diaphania nitidalis,Diaphorina citri, Diaprepes abbreviatus, Diatraea saccharalis,differential grasshopper, Ditylenchus spp., Dociostaurus maroccanus,Drosophila suzukii, Dryocosmus kuriphilus, Earias perhuegeli, Epicautavittata, Epilachna varivestis, Erionota thrax, Eriosomatinae, Euleiaheraclei, Eumetopina flavipes, Eupoecilia ambiguella, European cornborer, Eurydema oleracea, Eurygaster integriceps, forest bug,Frankliniella tritici, Galleria mellonella, garden dart, glassy-wingedsharpshooter, greenhouse whitefly, Gryllotalpa orientalis, Grylluspennsylvanicus, gypsy moths, Helicoverpa armigera, Helicoverpagelotopoeon, Helicoverpa punctigera, Helicoverpa zea, Heliothisvirescens, Henosepilachna vigintioctopunctata, Hessian fly, Heteroderaspp., Jacobiasca formosana, Japanese beetle, Khapra beetle, Lampidesboeticus, leaf miner, Lepidiota consobrina, Lepidosaphes beckii,Lepidosaphes ulmi, Leptoglossus zonatus, Leptopterna dolabrata, lesserwax moth, Leucoptera (moth), Leucoptera caffeina, light brown applemoth, Lissorhoptrus oryzophilus, long-tailed Skipper, Lygus, Lygushesperus, Maconellicoccus hirsutus, Macrodactylus subspinosus,Macrosiphum euphorbiae, maize weevil, Manduca sexta, Mayetiola hordei,Mealybug, Meloidogyne spp., Megacopta cribraria, Metcalfa pruinosa,moths, leek moth, Myzus persicae, Naccobus spp., Nezara viridula, oakprocessionary, olive fruit fly, Ophiomyia simplex, Opisina arenosella,Opomyza, Opomyza florum, Opomyzidae, Oscinella frit, Ostriniafurnacalis, Oxycarenus hyalinipennis, papaya mealy bug, Papiliodemodocus, Paratachardina pseudolobata, Pentatomoidea, Phthorimaeaoperculella, Phyllophaga, Phylloxera, Phylloxeridae, Phylloxeroidea,Pieris brassicae, pink bollworm, Planococcus citri, Platynotaidaeusalis, Plum curculio, Pratylenchus spp., Prionus californicus,Pseudococcus viburni, Pyralis farinalis, red imported fire ant, redlocust, root lesion nematodes, root knot nematodes, Radopholus spp.,Rotylenchulus spp., Rhagoletis cerasi, Rhagoletis indifferens,Rhagoletis mendax, Rhopalosiphum maidis, Rhyacionia frustrana,Rhynchophorus ferrugineus, Rhynchophorus palmarum, Rhyzopertha, ricemoth, rice stink bug, Russian wheat aphid, San Jose scale, scale insect,Schistocerca americana, Sciaridae, Scirtothrips dorsalis, Scutelleridae,Scutiphora pedicellata, seed gall nematodes, serpentine leaf miner,silverleaf whitefly, Sipha flava, small hive beetle, Southwestern cornborer, soybean aphid, Spodoptera cilium, Spodoptera litura, spottedcucumber beetle, squash vine borer, stem Nematodes, Stenotus binotatus,Strauzia longipennis, striped flea beetle, sunn pest, sweetpotato bug,tarnished plant bug, thrips, Thrips angusticeps, Thrips palmi, Toxopteracitricida, Trichodorus spp., Trioza erytreae, turnip moth, Tutaabsoluta, Tylenchulus spp., varied carpet beetle, Virachola isocrates,waxworm, Western corn rootworm, Western flower thrips, wheat fly, wheatweevil, whitefly, winter moth, and Xiphenema spp.

For example, the insect or worm pathogen can be army worm, blackcutworm, European corn borer, fall armyworm, cutworm, Japanese beetle,lesser cornstalk borer, maize billbug, seed corn maggot, webworm,southern cornstalk borer, southern corn rootworm, southern potatowireworm, stalk borer, sugarcane beetle, white grubs, cabbage looper,boll weevil, yellow striped armyworm, cereal leaf beetle, chinch bug,aphids, beet armyworm, Mexican bean beetle, soybean looper, soybean stemborer, or a combination thereof.

Proteins and Peptides that Enhance Stress Resistance in Plants

The invention also relates to fusion proteins comprising a targetingsequence, exosporium protein, or exosporium protein fragment and atleast one protein or peptide that enhances stress resistance in a plant.

For example, the protein or peptide that enhances stress resistance in aplant comprises an enzyme that degrades a stress-related compound.Stress-related compounds include, but are not limited to,aminocyclopropane-1-carboxylic acid (ACC), reactive oxygen species,nitric oxide, oxylipins, and phenolics. Specific reactive oxygen speciesinclude hydroxyl, hydrogen peroxide, oxygen, and superoxide. The enzymethat degrades a stress-related compound can comprise a superoxidedismutase, an oxidase, a catalase, an aminocyclopropane-1-carboxylicacid deaminase, a peroxidase, an antioxidant enzyme, or an antioxidantpeptide.

The protein or peptide that enhances stress resistance in a plant canalso comprise a protein or peptide that protects a plant from anenvironmental stress. The environmental stress can comprise, forexample, drought, flood, heat, freezing, salt, heavy metals, low pH,high pH, or a combination thereof. For instance, the protein or peptidethat protects a plant from an environmental stress can comprises an icenucleation protein, a prolinase, a phenylalanine ammonia lyase, anisochorismate synthase, an isochorismate pyruvate lyase, or a cholinedehydrogenase.

Plant Binding Proteins and Peptides

The invention also relates to fusion proteins comprising a targetingsequence, exosporium protein, or exosporium protein fragment and atleast plant binding protein or peptide. The plant binding protein orpeptide can be any protein or peptide that is capable of specifically ornon-specifically binding to any part of a plant (e.g., a plant root oran aerial portion of a plant such as a leaf, stem, flower, or fruit) orto plant matter. Thus, for example, the plant binding protein or peptidecan be a root binding protein or peptide, or a leaf binding protein orpeptide.

Suitable plant binding proteins and peptides include adhesins (e.g.,rhicadhesin), flagellins, omptins, lectins, expansins, biofilmstructural proteins (e.g., TasA or YuaB) pilus proteins, curlusproteins, intimins, invasins, agglutinins, and afimbrial proteins.

Other Fusion Proteins

The present invention further relates to fusion proteins comprising atleast one protein or peptide of interest and an exosporium proteincomprising an exosporium protein comprising an amino acid sequencehaving at least 85% identity with any one of SEQ ID NOs: 71, 75, 80, 81,82, 83, and 84. Alternatively, the exosporium protein can comprise anamino acid sequence having at least 90%, at least 95%, at least 98%, atleast 99%, or at least 100% identity with any one of SEQ ID NOs: 71, 75,80, 81, 82, 83, and 84.

The protein or peptide of interest can comprise any protein or peptide.For example, the protein or peptide of interest can comprise any of theproteins or peptides described herein. For example, the protein orpeptide of interest can comprise any of the plant growth stimulatingproteins or peptides described herein, any of the proteins or peptidesthat protect a plant from a pathogen described herein, any of theproteins or peptides that enhances stress resistance in a plantdescribed herein, or any of the a plant binding proteins or peptidesdescribed herein.

Thus, where the protein or peptide of interest comprises a plant growthstimulating protein or peptide, the plant growth stimulating protein orpeptide can comprise a peptide hormone, a non-hormone peptide, or anenzyme involved in the production or activation of a plant growthstimulating compound. Alternatively, the plant growth stimulatingprotein or peptide can comprise any of the enzymes that degrade ormodify a bacterial, fungal, or plant nutrient source describedhereinbelow.

Recombinant Bacillus cereus Family Members that Express the FusionProteins

The present invention also relates to a recombinant Bacillus cereusfamily member that expresses a fusion protein. The fusion protein can beany of the fusion proteins discussed above.

The recombinant Bacillus cereus family member can coexpress two or moreof any of the fusion proteins discussed above. For example, therecombinant Bacillus cereus family member can coexpress at least onefusion protein that comprises a plant binding protein or peptide,together with at least one fusion protein comprising a plant growthstimulating protein or peptide, at least one fusion protein comprising aprotein or peptide that protects a plant from a pathogen, or at leastone protein or peptide that enhances stress resistance in a plant.

The recombinant Bacillus cereus family member can comprise Bacillusanthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides,Bacillus pseudomycoides, Bacillus samanii, Bacillus gaemokensis,Bacillus weihenstephensis, or a combination thereof. For example, therecombinant Bacillus cereus family member can comprise Bacillus cereus,Bacillus thuringiensis, Bacillus pseudomycoides, or Bacillus mycoides.In particular, the recombinant Bacillus cereus family member cancomprise Bacillus thuringiensis or Bacillus mycoides.

To generate a recombinant Bacillus cereus family member expressing afusion protein, any Bacillus cereus family member can be conjugated,transduced, or transformed with a vector encoding the fusion proteinusing standard methods known in the art (e.g., by electroporation). Thebacteria can then be screened to identify transformants by any methodknown in the art. For example, where the vector includes an antibioticresistance gene, the bacteria can be screened for antibiotic resistance.Alternatively, DNA encoding the fusion protein can be integrated intothe chromosomal DNA of a B. cereus family member host. The recombinantBacillus cereus family member can then exposed to conditions which willinduce sporulation. Suitable conditions for inducing sporulation areknown in the art. For example, the recombinant Bacillus cereus familymember can be plated onto agar plates, and incubated at a temperature ofabout 30° C. for several days (e.g., 3 days).

Inactivated strains, non-toxic strains, or genetically manipulatedstrains of any of the above species can also suitably be used. Forexample, a Bacillus thuringiensis that lacks the Cry toxin can be used.Alternatively or in addition, once the recombinant B. cereus familyspores expressing the fusion protein have been generated, they can beinactivated to prevent further germination once in use. Any method forinactivating bacterial spores that is known in the art can be used.Suitable methods include, without limitation, heat treatment, gammairradiation, x-ray irradiation, UV-A irradiation, UV-B irradiation,chemical treatment (e.g., treatment with gluteraldehyde, formaldehyde,hydrogen peroxide, acetic acid, bleach, or any combination thereof), ora combination thereof. Alternatively, spores derived from nontoxigenicstrains, or genetically or physically inactivated strains, can be used.

Recombinant Bacillus cereus Family Members Having Plant-Growth PromotingEffects and/or Other Beneficial Attributes

Many Bacillus cereus family member strains have inherent beneficialattributes. For example, some strains have plant-growth promotingeffects. Any of the fusion proteins described herein can be expressed insuch strains.

For example, the recombinant Bacillus cereus family member can comprisea plant-growth promoting strain of bacteria.

The plant-growth promoting strain of bacteria can comprise a strain ofbacteria that produces an insecticidal toxin (e.g., a Cry toxin),produces a fungicidal compound (e.g., a β-1,3-glucanase, a chitosinase,a lyticase, or a combination thereof), produces a nematocidal compound(e.g., a Cry toxin), produces a bacteriocidal compound, is resistant toone or more antibiotics, comprises one or more freely replicatingplasmids, binds to plant roots, colonizes plant roots, forms biofilms,solubilizes nutrients, secretes organic acids, or any combinationthereof.

For example, where the recombinant Bacillus cereus family membercomprises a plant-growth promoting strain of bacteria, the plantgrowth-promoting strain of bacteria can comprise Bacillus mycoides BT155(NRRL No. B-50921), Bacillus mycoides EE118 (NRRL No. B-50918), Bacillusmycoides EE141 (NRRL No. B-50916), Bacillus mycoides BT46-3 (NRRL No.B-50922), Bacillus cereus family member EE128 (NRRL No. B-50917),Bacillus thuringiensis BT013A (NRRL No. B-50924), or Bacillus cereusfamily member EE349 (NRRL No. B-50928). Each of these strains wasdeposited with the United States Department of Agriculture (USDA)Agricultural Research Service (ARS), having the address 1815 NorthUniversity Street, Peoria, Ill. 61604 U.S.A., on Mar. 10, 2014, and isidentified by the NRRL deposit number provided in parentheses.

These plant-growth promoting strains were isolated from the rhizospheresof various vigorous plants and were identified by their 16S rRNAsequences (provided herein as SEQ ID NOs. 104-110), and throughbiochemical assays. The strains were identified at least to their genusdesignation by means of conventional biochemistry and morphologicalindicators. Biochemical assays for confirmed Gram-positive strains suchas Bacillus included growth on PEA medium and nutrient agar, microscopicexamination, growth on 5% and 7.5% NaCl medium, growth at pH 5 and pH 9,growth at 42° C. and 50° C., the ability to produce acid uponfermentation with cellobiose, lactose, glycerol, glucose, sucrose,d-mannitol, and starch; fluorescent pigment production; gelatinhydrolysis; nitrate reduction; catalase production, starch hydrolysis;oxidase reaction, urease production and motility. Identification ofthese strains and demonstration of their plant-growth promoting effectsare described further in the Examples hereinbelow.

For example, the recombinant Bacillus cereus family member comprising aplant-growth promoting strain of bacteria can comprise Bacillus mycoidesBT155, Bacillus mycoides EE141, or Bacillus thuringiensis BT013A. Therecombinant Bacillus cereus family member can express any of the fusionproteins described herein, e.g., a fusion protein comprising thetargeting sequence of SEQ ID NO: 60 and a non-hormone peptide (e.g.,kunitz trypsin inhibitor (KTI)), an enzyme involved in the production oractivation of a plant growth stimulating compound (e.g., a chitosinase),a plant binding protein or peptide (e.g., TasA); a protein or peptidethat protects a plant from a pathogen (e.g., TasA), or an enzyme thatdegrades or modifies a bacterial, fungal, or plant nutrient source(e.g., a phosphatase such as PhoA or phytase, or an endoglucanase).

Promoters

In any of the recombinant Bacillus cereus family members describedherein, the fusion protein can be expressed under the control of apromoter that is native to the targeting sequence, the exosporiumprotein, or the exosporium protein fragment of the fusion protein. Forexample, where the fusion protein comprises a targeting sequence derivedfrom B. anthracis Sterne BclA (e.g., amino acids 20-35 of SEQ ID NO: 1,amino acids 1-35 of SEQ ID NO: 1, SEQ ID NO: 1, or SEQ ID NO: 60) orwhere the fusion protein comprises full length BclA (SEQ ID NO: 2) or afragment of full length BclA (e.g., SEQ ID NO: 59), the fusion proteincan be expressed under the control of a promoter that is normallyassociated with the BclA gene in the genome of B. anthracis Sterne(e.g., the promoter of SEQ ID NO: 85).

Alternatively, the fusion protein can be expressed under the control ofa high-expression sporulation promoter. In some cases, the promoter thatis native to the targeting sequence, exosporium protein, or exosporiumprotein fragment will be a high-expression sporulation promoter. Inother cases, the promoter that is native to the targeting sequence,exosporium protein, or exosporium protein fragment will not be ahigh-expression sporulation promoter. In the latter cases, it may beadvantageous to replace the native promoter with a high-expressionsporulation promoter. Expression of the fusion protein under the controlof a high-expression sporulation promoter provides for increasedexpression of the fusion protein on the exosporium of the Bacilluscereus family member.

The high-expression sporulation promoter can comprise one or moresigma-K sporulation-specific polymerase promoter sequences.

Suitable high-expression sporulation promoters for use in expressing thefusion proteins in a Bacillus cereus family member include those listedin Table 2 below:

TABLE 2 Promoter Sequences Promoter (SEQ ID NO.) Sequence BclA promoterTAATCACCCTCTTCCAAATCAAT CATATGTTA TA CATATACTA AACT(B. anthracis Sterne) TTCCATTTTTTTAAATTGTTCAAGTAGTTTAAGATTTCTTTTCAATAAT(SEQ ID NO: 85) TCAAATGTCCGTGTCATTTTCTTTCGGTTTTG CATCTACTA TATAATGAACGCTTTATGGAGGTGAATTTATG BetA promoterATTTATTTCATTCAATTTTTCCTATTTAGTACCTACCGCACTCACAAAA (B. anthracis Sterne)AGCACCTCTCATTAATTTATATTATAGTCATTGAAATCTAATTTAATGA (SEQ ID NO: 86) AATCATCATACTATA TGTTTTATAAGAAGTAAAGGTAC CATACTTAATTAATACATATCTATACACTTCAATATCACAGCATGCAGTTGAATTATATCCAACTTTCATTTCAAATTAAATAAGTGCCTCCGCTATTGTGAATG TCATTTACTCTCCCTACTACATTTAATA ATTATGACAAGCAATCATAG GAGGTTACTACATG BAS1882 promoterAATTACATAACAAGAACTACATTAGGGAGCAAGCAGTCTAGCGAAAG (B. anthracis Sterne)CTAACTGCTTTTTTATTAAATAACTATTTTATTAAATTTCATATATACA (SEQ ID NO: 87)ATCGCTTGTCCATTTCATTTGGCTCTACCCACG CATTTACTA TTAGTAATATGAATTTTTCAGAGGTGGATTTTATT Gene 3572 promoterCTATGATTTAAGATACACAATAGCAAAAGAGAAA CATATTATA TAAC (B. weihenstephensisGATAAATGAAACTTATGTATATGTATGGTAACTGTATATATTACTACA KBAB 4)ATACAGTATACTCATAGGAGGTAGGTATG (SEQ ID NO: 88) YVTN β-propellerGGTAGGTAGATTTGAAATATGATGAAGAAAAGGAATAACTAAAAGGA protein promoterGTCGATATCCGACTCCTTTTAGTTATAAATAATGTGGAATTAGAGTAT (B. weihenstephensisAATTTTATATAGGTATATTGTATTAGATGAACGCTTTATCCTTTAATTG KBAB 4)TGATTAATGATGGATTGTAAGAGAAGGGGCTTACAGTCCTTTTTTTAT (SEQ ID NO: 89)GGTGTTCTATAAGCCTTTTTAAAAGGGGTACCACCCCACACCCAAAAACAGGGGGGGTTATAACTACATATTGGATGTTTTGTAACGTACAAGAATCGGTATTAATTACCCTGTAAATAAGTTATGTGTATATAAGGTAACTT T ATATATTCTCCTACAATAAAATAAAGGAGGTAATAAAGTG Cry1A promoterAACCCTTAATGCATTGGTTAAACATTGTAAAGTCTAAAGCATGGATAA (B. thuringiensisTGGGCGAGAAGTAAGTAGATTGTTAACACCCTGGGTCAAAAATTGAT HD-73)ATTTAGTAAAATTAGTTGCACTTTGTGCATTTTTT CATAAGATG AGT C (SEQ ID NO: 90)ATATGTTT TAAATTGTAGTAATGAAAAACAGTAT TATATCATAATGA ATTGGTATCTTAATAAAAGAGATGGAGGTAACTTA ExsY promoterTAATTCCACCTTCCCTTATCCTCTTTCGCCTATTTAAAAAAAGGTCTTG (B. thuringiensisAGATTGTGACCAAATCTCCTCAACTCC AATATCTTA TTAATGTAAATA serovar konkukianCAAACAAGAAGATAAGGAGTGACATTAA str. 97-27) (SEQ ID NO: 91) CotY promoterAGGATGTCTTTTTTTATATTGTATTATGTACATCCCTACTATATAAATT (B. thuringiensis A1CCCTGCTTTTATCGTAAGAATTAACGTAATATCAACCATATCCCGTT C Hakam) ATATTGTAGTAGTGTATGTCAGAACTCACGAGAAGGAGTGAACATAA (SEQ ID NO: 92) YjcA promoterTTAATGTCACTCCTTATCTTCTTGTTTGTATTTACATT AATAAGA TATT (B. thuringiensisGGAGTTGAGGAGATTTGGTCACAATCTCAAGACCTTTTTTTTAAATAG serovar kurstaki str.GCGAAAGAGGATAAGGGAAGGTGGAATTA HD73) (SEQ ID NO: 93) YjcB promoterATATATTTTCATAATACGAGAAAAAGCGGAGTTTAAAAGAATGAGGG (B. thuringiensisAACGGAAATAAAGAGTTGTT CATATAGTA AATAGACAGAATTGACAG serovar kurstaki str.TAGAGGAGA HD73) (SEQ ID NO: 94) BxpB promoterAAACTAAATAATGAGCTAAGCATGGATTGGGTGGCAGAATTATCTGC (B. thuringiensis A1CACCCAATC CATGCTTAA CGAGTATTATTATGTAAATTTCTTAAAATT Hakam)GGGAACTTGTCTAGAACATAGAACCTGTCCTTTT CATTAACTG AAAG (SEQ ID NO: 95)TAGAAACAGATAAAGGAGTGAAAAACA Rhamnose promoterATTCACTACAACGGGGATGAGTTTGATGCGGATA CATATGAGA AGTA (B. thuringiensis A1CCGGAAAGTGTTTGTAGAA CATTACAA AGATATATTATCTCCATCATA Hakam)AAGGAGAGATGCAAAG (SEQ ID NO: 96) CotY/CotZ promoterCGCGCACCACTTCGTCGTACAACAACGCAAGAAGAAGTTGGGGATAC (B. anthracis Sterne)AGCAGTATTCTTATTCAGTGATTTAGCACGCGGCGTAACAGGAGAAA (SEQ ID NO: 97)ACATTCACGTTGATTCAGGGTAT CATATCTTA GGATAAATATAATATTAATTTTAAAGGACAATCTCTACATGTTGAGATTGTCCTTTTTATTTGTTCTTAGAAAGAACGATTTTTAACGAAAGTTCTTACCACGTTATGAATATAAGTATAATAGTACACGATTTATTCAGCTACGTA BclC promoterTGAAGTATCTAGAGCTAATTTACGCAAAGGAATCTCAGGACAACACT (B. anthracis Sterne)TTCGCAACACCTATATTTTAAATTTAATAAAAAAAGAGACTCCGGAGT (SEQ ID NO: 98)CAGAAATTATAAAGCTAGCTGGGTTCAAATCAAAAATTTCACTAAAACGATATTATCAATACGCAGAAAATGGAAAAAACGCCTTATCATAAGGCGTTTTTTCCATTTTTTCTTCAAACAAACGATTTTACTATGACCATTTA ACTAATTTTTG CATCTACTATGATGAGTTTCATTCACATTCTCATTAG AAAGGAGAGATTTAATG Sigma K promoterTATATCATATGTAAAATTAGTTCTTATTCCCA CATATCATA TAGAATC (B. anthracis Sterne)GC CATATTATA CATGCAGAAAACTAAGTATGGTATTATTCTTAAATTG (SEQ ID NO: 99)TTTAGCACCTTCTAATATTACAGATAGAATCCGTCATTTTCAACAGTGAACATGGATTTCTTCTGAACACAACTCTTTTTCTTTCCTTATTTCCAAAAAGAAAAGCAGCCCATTTTAAAATACGGCTGCTTGTAATGTACATTA InhA promoterTATCACATAACTCTTTATTTTTAATATTTCGA CATAAAGTG AAACTTT (B. thuringiensis A1AATCAGTGGGGGCTTTGTTCATCCCCCCACTGATTATTAATTGAACCA Hakam )AGGGATAAAAAGATAGAGGGTCTGACCAGAAAACTGGAGGGCATGA (SEQ ID NO: 100)TTCTATAACAAAAAGCTTAATGTTTATAGAATTATGTCTTTTTATATAGGGAGGGTAGTAAACAGAGATTTGGACAAAAATGCACCGATTTATCTGAATTTTAAGTTTTATAAAGGGGAGAAATG BclA clusterATTTTTTACTTAGCAGTAAAACTGATATCAGTTTTACTGCTTTTTCATT glycosyl transferaseTTTAAATTCAATCATTAAATCTTCCTTTTCTACATAGT CATAATGTT GT operon 1ATGACATTCCGTAGGAGGCACTTATA (B. thuringiensis serovar konkukianstr. 97-27) (SEQ ID NO: 101) BclA clusterACATAAATTCACCTCCATAAAGCGTTCATTATATAGTAGATGCAAAAC glycosyl transferaseCGAAAGAAAATGACACGGACATTTGAATTATTGAAAAGAAATCTTAA operon 2ACTACTTGAACAATTTAAAAAAATGGAAAGTTTAGTATATGTATAA C (B. thuringiensisATATGATT GATTTGGAAGAGGGTGATTA serovar kurstaki str. HD73)(SEQ ID NO: 102) Glycosyl transferase TTCTATTTTCCAA CATAACATGCTACGATTAAATGGTTTTTTGCAAAT promoterGCCTTCTTGGGAAGAAGGATTAGAGCGTTTTTTTATAGAAACCAAAAG (B. thuringiensis A1TCATTAACAATTTTAAGTTAATGACTTTTTTGTTTGCCTTTAAGAGGTT Hakam)TTATGTTACTATAATTATAGTATCAGGTACTAATAACAAGTATAAGTA (SEQ ID NO: 103)TTTCTGGGAGGATATATCA

In the promoter sequences listed in Table 2 above, the locations of thesigma-K sporulation-specific polymerase promoter sequences are indicatedby bold and underlined text. The CrylA promoter (B. thuringiensis HD-73;SEQ ID NO: 90) has a total of four sigma-K sequences, two of whichoverlap with one another, as indicated by the double underlining inTable 2.

Preferred high-expression sporulation promoters for use in expressingthe fusion proteins in a Bacillus cereus family member include the BetApromoter (B. anthracis Sterne; SEQ ID NO: 86), the BclA promoter (B.anthracis Sterne; SEQ ID NO: 85), the BclA cluster glycosyl transferaseoperons 1 and 2 promoters (B. anthracis Sterne; SEQ ID NOs: 101 and102), and the YVTN β-propeller protein promoter (B. weihenstephensisKBAB 4; SEQ ID NO: 89).

In any of the recombinant Bacillus cereus family members describedherein, the fusion protein can be expressed under the control of asporulation promoter comprising a nucleic acid sequence having at least80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identity with a nucleic acid sequence of any one of SEQ ID NOs: 85-103.

When the sporulation promoter comprising a nucleic acid sequence havingat least 80%, at least 90%, at least 95%, at least 98%, or at least 99%identity with a nucleic acid sequence of any one of SEQ ID NOs: 85-103,the sigma-K sporulation-specific polymerase promoter sequence orsequences preferably have 100% identity with the correspondingnucleotides of SEQ ID NO: 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, or 103. For example, as illustrated inTable 2 above, the BclA promoter of B. anthracis Sterne (SEQ ID NO: 85)has sigma-K sporulation-specific polymerase promoter sequences atnucleotides 24-32, 35-43, and 129-137. Thus, if the sporulation promotercomprises a sequence having at least 90% identity with the nucleic acidsequence of SEQ ID NO: 85, it is preferred that the nucleotides of thesporulation promoter corresponding to nucleotides 24-32, 35-43, and129-137 of SEQ ID NO: 85 have 100% identity with nucleotides 24-32,35-43, and 129-137 of SEQ ID NO: 85.

Formulations

The present invention also relates to formulations comprising any of therecombinant Bacillus cereus family members discussed in the precedingsection and an agriculturally acceptable carrier.

The agriculturally acceptable carrier can be any carrier suitable foragricultural use. For example, suitable agriculturally acceptablecarriers include, but are not limited to dispersants, surfactants,additives, water, thickeners, anti-caking agents, residue breakdown,composting formulations, granular applications, diatomaceous earth,oils, coloring agents, stabilizers, preservatives, polymers, coatings,and combinations thereof.

The additive can comprise an oil, a gum, a resin, a clay, apolyoxyethylene glycol, a terpene, a viscid organic, a fatty acid ester,a sulfated alcohol, an alkyl sulfonate, a petroleum sulfonate, analcohol sulfate, a sodium alkyl butane diamate, a polyester of sodiumthiobutane dioate, a benzene acetonitrile derivative, a proteinaceousmaterial (e.g., a milk product, wheat flour, soybean meal, blood,albumin, gelatin, or a combination thereof), or a combination thereof.

The thickener can comprise a long chain alkylsulfonate of polyethyleneglycol, a polyoxyethylene oleate, or a combination thereof.

The surfactant can comprise a heavy petroleum oil, a heavy petroleumdistillate, a polyol fatty acid ester, a polyethoxylated fatty acidester, an aryl alkyl polyoxyethylene glycol, an alkyl amine acetate, analkyl aryl sulfonate, a polyhydric alcohol, an alkyl phosphate, or acombination thereof.

The anti-caking agent comprises a sodium salt, a calcium carbonate,diatomaceous earth, or a combination thereof. For example, the sodiumsalt can comprise a sodium salt of monomethyl naphthalene sulfonate, asodium salt of dimethyl naphthalene sulfonate, a sodium sulfite, asodium sulfate, or a combination thereof.

Suitable agriculturally acceptable carriers include vermiculite,charcoal, sugar factory carbonation press mud, rice husk, carboxymethylcellulose, peat, perlite, fine sand, calcium carbonate, flour, alum, astarch, talc, polyvinyl pyrrolidone, or a combination thereof.

The formulation can comprise a seed coating formulation, a liquidformulation for application to plants or to a plant growth medium, or asolid formulation for application to plants or to a plant growth medium.

For example, the seed coating formulation can comprise an aqueous oroil-based solution for application to seeds. Alternatively, the seedcoating formulation can comprise a powder or granular formulation forapplication to seeds.

The liquid formulation for application to plants or to a plant growthmedium can comprise a concentrated formulation or a ready-to-useformulation.

The solid formulation for application to plants or to a plant growthmedium can comprises a granular formulation or a powder agent.

Any of the above formulations can also comprise an agrochemical, forexample, a fertilizer, a micronutrient fertilizer material, aninsecticide, a herbicide, a plant growth amendment, a fungicide, aninsecticide, a molluscicide, an algicide, a bacterial inoculant, afungal inoculant, or a combination thereof.

The fertilizer can comprise a liquid fertilizer.

The fertilizer can comprise ammonium sulfate, ammonium nitrate, ammoniumsulfate nitrate, ammonium chloride, ammonium bisulfate, ammoniumpolysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia,ammonium polyphosphate, aluminum sulfate, calcium nitrate, calciumammonium nitrate, calcium sulfate, calcined magnesite, calciticlimestone, calcium oxide, calcium nitrate, dolomitic limestone, hydratedlime, calcium carbonate, diammonium phosphate, monoammonium phosphate,magnesium nitrate, magnesium sulfate, potassium nitrate, potassiumchloride, potassium magnesium sulfate, potassium sulfate, sodiumnitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, ureaammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylidenediurea, K₂SO₄-2MgSO₄, kainite, sylvinite, kieserite, Epsom salts,elemental sulfur, marl, ground oyster shells, fish meal, oil cakes, fishmanure, blood meal, rock phosphate, super phosphates, slag, bone meal,wood ash, manure, bat guano, peat moss, compost, green sand, cottonseedmeal, feather meal, crab meal, fish emulsion, humic acid, or acombination thereof.

The micronutrient fertilizer material can comprise boric acid, a borate,a boron frit, copper sulfate, a copper frit, a copper chelate, a sodiumtetraborate decahydrate, an iron sulfate, an iron oxide, iron ammoniumsulfate, an iron frit, an iron chelate, a manganese sulfate, a manganeseoxide, a manganese chelate, a manganese chloride, a manganese frit, asodium molybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinccarbonate, a zinc frit, zinc phosphate, a zinc chelate, or a combinationthereof.

The insecticide can comprise an organophosphate, a carbamate, apyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, afluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester,a biologically-based insecticide, or a combination thereof.

The herbicide can comprise a chlorophenoxy compound, a nitrophenoliccompound, a nitrocresolic compound, a dipyridyl compound, an acetamide,an aliphatic acid, an anilide, a benzamide, a benzoic acid, a benzoicacid derivative, anisic acid, an anisic acid derivative, a benzonitrile,benzothiadiazinone dioxide, a thiocarbamate, a carbamate, a carbanilate,chloropyridinyl, a cyclohexenone derivative, a dinitroaminobenzenederivative, a fluorodinitrotoluidine compound, isoxazolidinone,nicotinic acid, isopropylamine, an isopropylamine derivatives,oxadiazolinone, a phosphate, a phthalate, a picolinic acid compound, atriazine, a triazole, a uracil, a urea derivative, endothall, sodiumchlorate, or a combination thereof.

The fungicide can comprise a substituted benzene, a thiocarbamate, anethylene bis dithiocarbamate, a thiophthalidamide, a copper compound, anorganomercury compound, an organotin compound, a cadmium compound,anilazine, benomyl, cyclohexamide, dodine, etridiazole, iprodione,metlaxyl, thiamimefon, triforine, or a combination thereof.

The fungal inoculant can comprise a fungal inoculant of the familyGlomeraceae, a fungal inoculant of the family Claroidoglomeraceae, afungal inoculant of the family Gigasporaceae, a fungal inoculant of thefamily Acaulosporaceae, a fungal inoculant of the familySacculosporaceae, a fungal inoculant of the family Entrophosporaceae, afungal inoculant of the family Pacidsporaceae, a fungal inoculant of thefamily Diversisporaceae, a fungal inoculant of the familyParaglomeraceae, a fungal inoculant of the family Archaeosporaceae, afungal inoculant of the family Geosiphonaceae, a fungal inoculant of thefamily Ambisporaceae, a fungal inoculant of the familyScutellosporaceae, a fungal inoculant of the family Dentiscultataceae, afungal inoculant of the family Racocetraceae, a fungal inoculant of thephylum Basidiomycota, a fungal inoculant of the phylum Ascomycota, afungal inoculant of the phylum Zygomycota, or a combination thereof.

The bacterial inoculant can comprise a bacterial inoculant of the genusRhizobium, a bacterial inoculant of the genus Bradyrhizobium, abacterial inoculant of the genus Mesorhizobium, a bacterial inoculant ofthe genus Azorhizobium, a bacterial inoculant of the genusAllorhizobium, a bacterial inoculant of the genus Sinorhizobium, abacterial inoculant of the genus Kluyvera, a bacterial inoculant of thegenus Azotobacter, a bacterial inoculant of the genus Pseudomonas, abacterial inoculant of the genus Azospirillium, a bacterial inoculant ofthe genus Bacillus, a bacterial inoculant of the genus Streptomyces, abacterial inoculant of the genus Paenibacillus, a bacterial inoculant ofthe genus Paracoccus, a bacterial inoculant of the genus Enterobacter, abacterial inoculant of the genus Alcaligenes, a bacterial inoculant ofthe genus Mycobacterium, a bacterial inoculant of the genus Trichoderma,a bacterial inoculant of the genus Gliocladium, a bacterial inoculant ofthe genus Glomus, a bacterial inoculant of the genus Klebsiella, or acombination thereof.

The bacterial inoculant can comprise a plant-growth promoting strain ofbacteria. The plant-growth promoting strain of bacteria can comprise astrain of bacteria that produces an insecticidal toxin (e.g., a Crytoxin), produces a fungicidal compound (e.g., a β-1,3-glucanase, achitosinase, a lyticase, or a combination thereof), produces anematocidal compound (e.g., a Cry toxin), produces a bacteriocidalcompound, is resistant to one or more antibiotics, comprises one or morefreely replicating plasmids, binds to plant roots, colonizes plantroots, forms biofilms, solubilizes nutrients, secretes organic acids, orany combination thereof.

For example, the bacterial inoculant can comprise Bacillus aryabhattaiCAP53 (NRRL No. B-50819), Bacillus aryabhattai CAP56 (NRRL No. B-50817),Bacillus flexus BT054 (NRRL No. B-50816), Paracoccus kondratievae NC35(NRRL No. B-50820), Bacillus mycoides BT155 (NRRL No. B-50921),Enterobacter cloacae CAP12 (NRRL No. B-50822), Bacillus nealsonii BOBA57(NRRL No. NRRL B-50821), Bacillus mycoides EE118 (NRRL No. B-50918),Bacillus subtilis EE148 (NRRL No. B-50927), Alcaligenes faecalis EE107(NRRL No. B-50920), Bacillus mycoides EE141 (NRRL NO. B-50916), Bacillusmycoides BT46-3 (NRRL No. B-50922), Bacillus cereus family member EE128(NRRL No. B-50917), Bacillus thuringiensis BT013A (NRRL No. B-50924),Paenibacillus massiliensis BT23 (NRRL No. B-50923), Bacillus cereusfamily member EE349 (NRRL No. B-50928), Bacillus subtilis EE218 (NRRLNo. B-50926), Bacillus megaterium EE281 (NRRL No. B-50925), or acombination thereof. Each of these strains was deposited with the UnitedStates Department of Agriculture (USDA) Agricultural Research Service(ARS), having the address 1815 North University Street, Peoria, Ill.61604 U.S.A., on Mar. 7, 2013 (Bacillus aryabhattai CAP53, Bacillusaryabhattai CAP56, Bacillus flexus BT054, Paracoccus kondratievae NC35,Enterobacter cloacae CAP12, and Bacillus nealsonii BOBA57) or on Mar.10, 2014 (Bacillus mycoides BT155, Bacillus mycoides EE118, Bacillussubtilis EE148, Alcaligenes faecalis EE107, Bacillus mycoides EE141,Bacillus mycoides BT46-3, Bacillus cereus family member EE128, Bacillusthuringiensis BT013A, Paenibacillus massiliensis BT23, Bacillus cereusfamily member EE349, Bacillus subtilis EE218, and Bacillus megateriumEE281), and is identified by the NRRL numbers provided in parentheses.

These plant-growth promoting strains were isolated from the rhizospheresof various vigorous plants and were identified by their 16S rRNAsequences (provided herein as SEQ ID NOs. 104-121), and throughbiochemical assays. The strains were identified at least to their genusdesignation by means of conventional biochemistry and morphologicalindicators. Biochemical assays for confirmed Gram-negative strains suchas Paracoccus kondratievae, Alcaligenes faecalis, and Enterobactercloacae included growth on MacConkey medium and nutrient agar,microscopic examination, growth on 5% and 7.5% NaCl medium, growth at pH5 and pH 9, growth at 42° C. and 50° C., the ability to produce acidupon fermentation with cellobiose, lactose, glycerol, glucose, sucrose,d-mannitol, and starch; fluorescent pigment production; gelatinhydrolysis; nitrate reduction; starch hydrolysis; oxidase reaction,catalase production, urease production and motility. Similarly, thebiochemical assays for confirmed Gram-positive strains such as Bacillusand Paenibacillus included growth on PEA medium and nutrient agar,microscopic examination, growth on 5% and 7.5% NaCl medium, growth at pH5 and pH 9, growth at 42° C. and 50° C., the ability to produce acidupon fermentation with cellobiose, lactose, glycerol, glucose, sucrose,d-mannitol, and starch; fluorescent pigment production; gelatinhydrolysis; nitrate reduction; catalase production, starch hydrolysis;oxidase reaction, urease production and motility. Identification ofthese strains and demonstration of their plant-growth promoting effectsare described further in the Examples hereinbelow.

For example, the formulation can comprise a plant-growth promotingstrain of bacteria comprising Paracoccus kondratievae NC35, Bacillusaryabhattai CAP53, or Bacillus megaterium EE281, wherein the formulationfurther comprises any of the recombinant Bacillus cereus family membersdescribed herein, including any of the recombinant plant-growthpromoting Bacillus cereus family member strains herein (e.g.,recombinant Bacillus mycoides BT155, Bacillus mycoides EE141, orBacillus thuringiensis BT013A). The recombinant plant-growth promotingBacillus cereus family member strain can express any of the fusionproteins described herein, e.g., a fusion protein comprising thetargeting sequence of SEQ ID NO: 60 and a non-hormone peptide (e.g.,kunitz trypsin inhibitor (KTI)), an enzyme involved in the production oractivation of a plant growth stimulating compound (e.g., a chitosinase),a plant binding protein or peptide (e.g., TasA); a protein or peptidethat protects a plant from a pathogen (e.g., TasA), or an enzyme thatdegrades or modifies a bacterial, fungal, or plant nutrient source(e.g., a phosphatase such as PhoA or phytase, or an endoglucanase).

Methods for Promoting Plant Growth

The present invention also relates to methods for stimulating plantgrowth. The method for stimulating plant growth comprises introducinginto a plant growth medium any of the recombinant Bacillus cereus familymembers discussed above or any of the formulations discussed above.Alternatively, any of the recombinant Bacillus cereus family membersdiscussed above or any of the formulations discussed above can beapplied to a plant, to a plant seed, or to an area surrounding a plantor a plant seed. In such methods, the plant growth stimulating proteinor peptide is physically attached to the exosporium of the recombinantBacillus family member.

Alternatively, the method for stimulating plant growth comprisesintroducing a recombinant Bacillus cereus family member expressing afusion protein into a plant growth medium or applying a recombinantBacillus cereus family member expressing a fusion protein to plant, aplant seed, or an area surrounding a plant or a plant seed. The fusionprotein comprises at least one plant growth stimulating protein orpeptide and a targeting sequence, exosporium protein, or exosporiumprotein fragment. The plant growth stimulating protein or peptide isphysically attached to the exosporium of the recombinant Bacillus familymember. The targeting sequence, exosporium protein, or exosporiumprotein fragment can be any of the targeting sequences, exosporiumproteins, or exosporium protein fragments listed above in paragraph[0005].

Furthermore, the targeting sequence can consist of 16 amino acids andhave at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 54%.Alternatively, the targeting sequence can consist of amino acids 1-35 ofSEQ ID NO: 1, amino acids 20-35 of SEQ ID NO: 1; SEQ ID NO: 1, or SEQ IDNO: 60.

The targeting sequence can comprise an amino acid sequence having atleast about 50% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 63%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 50% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 63%.

The targeting sequence can comprise an amino acid sequence having atleast about 50% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 72%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 50% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 72%.

The targeting sequence can comprises an amino acid sequence having atleast about 56% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 63%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 56% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 63%.

The targeting sequence can comprise an amino sequence having at leastabout 62% identity with amino acids 20-35 of SEQ ID NO: 1, wherein theidentity with amino acids 25-35 is at least about 72%. Alternatively,the targeting sequence consists of an amino acid sequence consisting of16 amino acids and having at least about 62% identity with amino acids20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 ofSEQ ID NO:1 is at least about 72%.

The targeting sequence can comprise an amino acid sequence having atleast about 68% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 81%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 68% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 81%.

The targeting sequence can comprise an amino sequence having at leastabout 75% identity with amino acids 20-35 of SEQ ID NO: 1, wherein theidentity with amino acids 25-35 is at least about 72%. Alternatively,the targeting sequence consists of an amino acid sequence consisting of16 amino acids and having at least about 75% identity with amino acids20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 ofSEQ ID NO:1 is at least about 72%.

The targeting sequence can comprise an amino sequence having at leastabout 75% identity with amino acids 20-35 of SEQ ID NO: 1, wherein theidentity with amino acids 25-35 is at least about 81%. Alternatively,the targeting sequence consists of an amino acid sequence consisting of16 amino acids and having at least about 75% identity with amino acids20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 ofSEQ ID NO:1 is at least about 81%.

The targeting sequence can comprise an amino acid sequence having atleast about 81% identity with amino acids 20-35 of SEQ ID NO:1, whereinthe identity with amino acids 25-35 is at least about 81%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 81% identity withamino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids25-35 is at least about 81%.

The targeting sequence can comprise an amino acid sequence having atleast about 81% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 90%.Alternatively, the targeting sequence consists of an amino acid sequenceconsisting of 16 amino acids and having at least about 81% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 90%.

Alternatively, the exosporium protein or exosporium protein fragment cancomprise an amino acid sequence having at least 90%, at least 95%, atleast 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46,48, 50, 52, 54, 56, 58, 59, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, and 84.

The plant growth stimulating protein can comprise an enzyme. Forexample, the enzyme can comprise an enzyme that degrades or modifies abacterial, fungal, or plant nutrient source. Such enzymes includecellulases, lipases, lignin oxidases, proteases, glycoside hydrolases,phosphatases, nitrogenases, nucleases, amidases, nitrate reductases,nitrite reductases, amylases, ammonia oxidases, ligninases,glucosidases, phospholipases, phytases, pectinases, glucanases,sulfatases, ureases, xylanases, and siderophores. When introduced into aplant growth medium or applied to a plant, seed, or an area surroundinga plant or a plant seed, fusion proteins comprising enzymes that degradeor modify a bacterial, fungal, or plant nutrient source can aid in theprocessing of nutrients in the vicinity of the plant and result inenhanced uptake of nutrients by the plant or by beneficial bacteria orfungi in the vicinity of the plant.

Suitable cellulases include endocellulases (e.g., an endogluconase suchas a Bacillus subtilis endoglucanase, a Bacillus thuringiensisendoglucanase, a Bacillus cereus endoglucanase, or a Bacillus clausiiendoglucanase), exocellulases (e.g., a Trichoderma reesei exocellulase),and β-glucosidases (e.g., a Bacillus subtilis β-glucosidase, a Bacillusthuringiensis β-glucosidase, a Bacillus cereus β-glucosidase, or aBacillus clausii B-glucosidase).

The lipase can comprise a Bacillus subtilis lipase, a Bacillusthuringiensis lipase, a Bacillus cereus lipase, or a Bacillus clausiilipase.

Suitable lignin oxidases comprise lignin peroxidases, laccases, glyoxaloxidases, ligninases, and manganese peroxidases.

The protease can comprise a subtilisin, an acid protease, an alkalineprotease, a proteinase, a peptidase, an endopeptidase, an exopeptidase,a thermolysin, a papain, a pepsin, a trypsin, a pronase, a carboxylase,a serine protease, a glutamic protease, an aspartate protease, acysteine protease, a threonine protease, or a metalloprotease.

The phosphatase can comprise a phosphoric monoester hydrolase, aphosphomonoesterase (e.g., PhoA4), a phosphoric diester hydrolase, aphosphodiesterase, a triphosphoric monoester hydrolase, a phosphorylanhydride hydrolase, a pyrophosphatase, a phytase (e.g., Bacillussubtilis EE148 phytase or Bacillus thuringiensis BT013A phytase), atrimetaphosphatase, or a triphosphatase.

The nitrogenase can comprise a Nif family nitrogenase (e.g.,Paenibacillus massiliensis NifBDEHKNXV).

In any of the above methods for stimulating plant growth, plants grownin the plant growth medium comprising the recombinant Bacillus cereusfamily member exhibit increased growth as compared to the growth ofplants in the identical plant growth medium that does not contain therecombinant Bacillus cereus family member.

In any of the above methods for stimulating plant growth, therecombinant Bacillus cereus family member can comprise any of therecombinant plant-growth promoting strains of bacteria described above.

In any of the above methods for stimulating plant growth, the fusionprotein can be expressed under the control of any of the promotersdescribed above.

Methods for Protecting a Plant from a Pathogen

The present invention further relates to methods for protecting a plantfrom a pathogen. Such methods comprise introducing any of therecombinant Bacillus cereus family members discussed above or any of theformulations discussed above into a plant growth medium. Alternatively,such methods comprise applying any of the recombinant Bacillus cereusfamily members discussed above or any of the formulations discussedabove to a plant, to a plant seed, or to an area surrounding a plant ora plant seed. In these methods, the protein or peptide that protects aplant from a pathogen is physically attached to the exosporium of therecombinant Bacillus cereus family member.

Plants grown in the plant growth medium comprising the recombinantBacillus cereus family member are less susceptible to infection with thepathogen as compared to plants grown in the identical plant growthmedium that does not contain the recombinant Bacillus cereus familymember. The reduced susceptibility the pathogen can be a result ofstimulation of the plant's immune system by the protein or peptide thatprotects a plant from a pathogen, or can result from a direct orindirect effect of the protein or peptide that protects a plant from apathogen on the pathogen.

Methods for Enhancing Stress Resistance in a Plant

The present invention further relates to methods for enhancing stressresistance in a plant. Such methods comprise introducing any of therecombinant Bacillus cereus family members discussed above or any of theformulations discussed above into a plant growth medium. Alternatively,such methods comprise applying any of the recombinant Bacillus cereusfamily members discussed above or any of the formulations discussedabove to a plant, to a plant seed, or to an area surrounding a plant ora plant seed. In these methods, the protein or peptide that enhancesstress resistance in a plant is physically attached to the exosporium ofthe recombinant Bacillus cereus family member.

Plants grown in the plant growth medium comprising the recombinantBacillus cereus family member are less susceptible to stress as comparedto plants grown in the identical plant growth medium that does notcontain the recombinant Bacillus cereus family member.

Methods for Immobilizing Bacillus cereus Family Member Spores on a Plant

The present invention is also directed to methods for immobilizing arecombinant Bacillus cereus family member spore on a plant. Thesemethods comprise introducing any of the recombinant Bacillus cereusfamily members discussed above or any of the formulations discussedabove into a plant growth medium. Alternatively, such methods compriseapplying any of the recombinant Bacillus cereus family members discussedabove or any of the formulations discussed above to a plant, to a plantseed, or to an area surrounding a plant or a plant seed. The plantbinding protein or peptide is physically attached to the exosporium ofthe recombinant Bacillus family member.

These methods allow the Bacillus cereus family member spore to bind to aplant, such that the spore is maintained on the plant. For example,these methods allow the Bacillus cereus family member spore to bind to aroot of a plant or to an aerial portion of a plant (e.g., foliage,stems, fruits, or flowers), such that the spore is maintained at theplant's root structure or on the aerial portion of a plant instead ofdissipating into the plant growth medium or into the environmentsurrounding the aerial portion of the plant.

In any of the methods for immobilizing a recombinant Bacillus cereusfamily member spore on a plant, the plant binding protein or peptide canselectively target and maintain the Bacillus cereus family member on theplant or on at plant structure or substructure (e.g., at plant roots andsubstructures of plant roots or at an aerial portion of a plant or asubstructure of an aerial portion of a plant).

Plant Growth Medium

In any of the above methods, the plant growth medium is material that iscapable of supporting the growth of a plant. The plant growth medium cancomprise soil, water, an aqueous solution, sand, gravel, apolysaccharide, mulch, compost, peat moss, straw, logs, clay, soybeanmeal, yeast extract, or a combination thereof. For example, the plantgrowth medium comprises soil, compost, peat moss, or a combinationthereof.

The plant growth medium can optionally be supplemented with a substratefor an enzyme. For example, the substrate can comprise tryptophan, anadenosine monophosphate, an adenosine diphosphate, an adenosinetriphosphate (e.g., adenosine-3-triphosphate), indole, atrimetaphosphate, ferrodoxin, acetoin, diacetyl, pyruvate, acetolactate,pectin, cellulose, methylcellulose, starch, chitin, pectin, a proteinmeal, a cellulose derivative, a phosphate, acetoin, chitosan, aninactive derivative of indole-3-acetic acid, an inactive derivative ofgibberellic acid, a xylan, choline, a choline derivative, proline, apolyproline, a proline rich meal, a proline-rich protein, phenylalanine,chorismate, an arabinoxylan, a fat, a wax, an oil, a phytic acid, alignin, a humic acid, choline, a choline derivative, or a combinationthereof.

Application Methods

In any of the above methods, the recombinant Bacillus cereus familymember or formulation can be introduced into the plant growth medium orapplied to a plant, to a plant seed, or to an area surrounding a plantor a plant seed.

For example, the method can comprise coating seeds with the recombinantBacillus cereus family member or a formulation containing therecombinant Bacillus cereus family member prior to planting.

Alternatively, the method can comprise applying the recombinant Bacilluscereus family member or formulation to an aerial portion of a plant,e.g., to foliage, stems, fruits, or flowers. For example, therecombinant Bacillus cereus family member or formulation can be sprayed,brushed, dipped, or otherwise applied to the leaves or other aerialportions of a plant.

The method can comprise introducing the recombinant Bacillus cereusfamily member into the plant growth medium by applying a liquid or solidformulation containing the recombinant Bacillus cereus family member tothe medium (e.g., soil, compost, peat moss, or a combination thereof).

The formulation can be applied to the plant growth medium prior to,concurrently with, or after planting of seeds, seedlings, cuttings,bulbs, or plants in the plant growth medium.

Co-Application of Agrochemicals

Any of the above methods can further comprise introducing at least oneagrochemical into the plant growth medium or applying at least oneagrochemical to plants or seeds. The agrochemical can be any of thoselisted above for inclusion in the formulations, or any combinationthereof.

Plants

The above methods can be practiced with a variety of plants. Forexample, the plant can be a dicotyledon, a monocotyledon, or agymnosperm.

For example, where the plant is a dicotyledon, the dicotyledon can beselected from the group consisting of bean, pea, tomato, pepper, squash,alfalfa, almond, aniseseed, apple, apricot, arracha, artichoke, avocado,bambara groundnut, beet, bergamot, black pepper, black wattle,blackberry, blueberry, bitter orange, bok-choi, Brazil nut, breadfruit,broccoli, broad bean, Brussels sprouts, buckwheat, cabbage, camelina,Chinese cabbage, cacao, cantaloupe, caraway seeds, cardoon, carob,carrot, cashew nuts, cassava, castor bean, cauliflower, celeriac,celery, cherry, chestnut, chickpea, chicory, chili pepper,chrysanthemum, cinnamon, citron, clementine, clove, clover, coffee, colanut, colza, corn, cotton, cottonseed, cowpea, crambe, cranberry, cress,cucumber, currant, custard apple, drumstick tree, earth pea, eggplant,endive, fennel, fenugreek, fig, filbert, flax, geranium, gooseberry,gourd, grape, grapefruit, guava, hemp, hempseed, henna, hop, horse bean,horseradish, indigo, jasmine, Jerusalem artichoke, jute, kale, kapok,kenaf, kohlrabi, kumquat, lavender, lemon, lentil, lespedeza, lettuce,lime, liquorice, litchi, loquat, lupine, macadamia nut, mace, mandarin,mangel, mango, medlar, melon, mint, mulberry, mustard, nectarine, nigerseed, nutmeg, okra, olive, opium, orange, papaya, parsnip, pea, peach,peanut, pear, pecan nut, persimmon, pigeon pea, pistachio nut, plantain,plum, pomegranate, pomelo, poppy seed, potato, sweet potato, prune,pumpkin, quebracho, quince, trees of the genus Cinchona, quinoa, radish,ramie, rapeseed, raspberry, rhea, rhubarb, rose, rubber, rutabaga,safflower, sainfoin, salsify, sapodilla, Satsuma, scorzonera, sesame,shea tree, soybean, spinach, squash, strawberry, sugar beet, sugarcane,sunflower, swede, sweet pepper, tangerine, tea, teff, tobacco, tomato,trefoil, tung tree, turnip, urena, vetch, walnut, watermelon, yerbamate, wintercress, shepherd's purse, garden cress, peppercress,watercress, pennycress, star anise, laurel, bay laurel, cassia, jamun,dill, tamarind, peppermint, oregano, rosemary, sage, soursop, pennywort,calophyllum, balsam pear, kukui nut, Tahitian chestnut, basil,huckleberry, hibiscus, passionfruit, star apple, sassafras, cactus, St.John's wort, loosestrife, hawthorn, cilantro, curry plant, kiwi, thyme,zucchini, ulluco, jicama, waterleaf, spiny monkey orange, yellow mombin,starfruit, amaranth, wasabi, Japanese pepper, yellow plum, mashua,Chinese toon, New Zealand spinach, bower spinach, ugu, tansy, chickweed,jocote, Malay apple, paracress, sowthistle, Chinese potato, horseparsley, hedge mustard, campion, agate, cassod tree, thistle, burnet,star gooseberry, saltwort, glasswort, sorrel, silver lace fern, collardgreens, primrose, cowslip, purslane, knotgrass, terebinth, tree lettuce,wild betel, West African pepper, yerba santa, tarragon, parsley,chervil, land cress, burnet saxifrage, honeyherb, butterbur, shiso,water pepper, perilla, bitter bean, oca, kampong, Chinese celery, lemonbasil, Thai basil, water mimosa, cicely, cabbage-tree, moringa, mauka,ostrich fern, rice paddy herb, yellow sawah lettuce, lovage, peppergrass, maca, bottle gourd, hyacinth bean, water spinach, catsear,fishwort, Okinawan spinach, lotus sweetjuice, gallant soldier, culantro,arugula, cardoon, caigua, mitsuba, chipilin, samphire, mampat, ebolo,ivy gourd, cabbage thistle, sea kale, chaya, huauzontle, Ethiopianmustard, magenta spreen, good king henry, epazole, lamb's quarters,centella plumed cockscomb, caper, rapini, napa cabbage, mizuna, Chinesesavoy, kai-lan, mustard greens, Malabar spinach, chard, marshmallow,climbing wattle, China jute, paprika, annatto seed, spearmint, savory,marjoram, cumin, chamomile, lemon balm, allspice, bilberry, cherimoya,cloudberry, damson, pitaya, durian, elderberry, feijoa, jackfruit,jambul, jujube, physalis, purple mangosteen, rambutan, redcurrant,blackcurrant, salal berry, satsuma, ugli fruit, azuki bean, black bean,black-eyed pea, borlotti bean, common bean, green bean, kidney bean,lima bean, mung bean, navy bean, pinto bean, runner bean, mangetout,snap pea, broccoflower, calabrese, nettle, bell pepper, raddichio,daikon, white radish, skirret, tat soi, broccolini, black radish,burdock root, fava bean, broccoli raab, lablab, lupin, sterculia, velvetbeans, winged beans, yam beans, mulga, ironweed, umbrella bush,tjuntjula, wakalpulka, witchetty bush, wiry wattle, chia, beech nut,candlenut, colocynth, mamoncillo, Maya nut, mongongo, ogbono nut,paradise nut, and cempedak.

Alternatively, the dicotyledon can be from a family selected from thegroup consisting of Acanthaceae (acanthus), Aceraceae (maple),Achariaceae, Achatocarpaceae (achatocarpus), Actinidiaceae (Chinesegooseberry), Adoxaceae (moschatel), Aextoxicaceae, Aizoaceae (figmarigold), Akaniaceae, Alangiaceae, Alseuosmiaceae, Alzateaceae,Amaranthaceae (amaranth), Amborellaceae, Anacardiaceae (sumac),Ancistrocladaceae, Anisophylleaceae, Annonaceae (custard apple),Apiaceae (carrot), Apocynaceae (dogbane), Aquifoliaceae (holly),Araliaceae (ginseng), Aristolochiaceae (birthwort), Asclepiadaceae(milkweed), Asteraceae (aster), Austrobaileyaceae, Balanopaceae,Balanophoraceae (balanophora), Balsaminaceae (touch-me-not),Barbeyaceae, Barclayaceae, Basellaceae (basella), Bataceae (saltwort),Begoniaceae (begonia), Berberidaceae (barberry), Betulaceae (birch),Bignoniaceae (trumpet creeper), Bixaceae (lipstick tree), Bombacaceae(kapok tree), Boraginaceae (borage), Brassicaceae (mustard, alsoCruciferae), Bretschneideraceae, Brunelliaceae (brunellia), Bruniaceae,Brunoniaceae, Buddlejaceae (butterfly bush), Burseraceae (frankincense),Buxaceae (boxwood), Byblidaceae, Cabombaceae (water shield), Cactaceae(cactus), Caesalpiniaceae, Callitrichaceae (water starwort),Calycanthaceae (strawberry shrub), Calyceraceae (calycera),Campanulaceae (bellflower), Canellaceae (canella), Cannabaceae (hemp),Capparaceae (caper), Caprifoliaceae (honeysuckle), Cardiopteridaceae,Caricaceae (papaya), Caryocaraceae (souari), Caryophyllaceae (pink),Casuarinaceae (she-oak), Cecropiaceae (cecropia), Celastraceae(bittersweet), Cephalotaceae, Ceratophyllaceae (hornwort),Cercidiphyllaceae (katsura tree), Chenopodiaceae (goosefoot),Chloranthaceae (chloranthus), Chrysobalanaceae (cocoa plum),Circaeasteraceae, Cistaceae (rockrose), Clethraceae (clethra),Clusiaceae (mangosteen, also Guttiferae), Cneoraceae, Columelliaceae,Combretaceae (Indian almond), Compositae (aster), Connaraceae(cannarus), Convolvulaceae (morning glory), Coriariaceae, Cornaceae(dogwood), Corynocarpaceae (karaka), Crassulaceae (stonecrop),Crossosomataceae (crossosoma), Crypteroniaceae, Cucurbitaceae(cucumber), Cunoniaceae (cunonia), Cuscutaceae (dodder), Cyrillaceae(cyrilla), Daphniphyllaceae, Datiscaceae (datisca), Davidsoniaceae,Degeneriaceae, Dialypetalanthaceae, Diapensiaceae (diapensia),Dichapetalaceae, Didiereaceae, Didymelaceae, Dilleniaceae (dillenia),Dioncophyllaceae, Dipentodontaceae, Dipsacaceae (teasel),Dipterocarpaceae (meranti), Donatiaceae, Droseraceae (sundew),Duckeodendraceae, Ebenaceae (ebony), Elaeagnaceae (oleaster),Elaeocarpaceae (elaeocarpus), Elatinaceae (waterwort), Empetraceae(crowberry), Epacridaceae (epacris), Eremolepidaceae (catkin-mistletoe),Ericaceae (heath), Erythroxylaceae (coca), Eucommiaceae, Eucryphiaceae,Euphorbiaceae (spurge), Eupomatiaceae, Eupteleaceae, Fabaceae (pea orlegume), Fagaceae (beech), Flacourtiaceae (flacourtia), Fouquieriaceae(ocotillo), Frankeniaceae (frankenia), Fumariaceae (fumitory),Garryaceae (silk tassel), Geissolomataceae, Gentianaceae (gentian),Geraniaceae (geranium), Gesneriaceae (gesneriad), Globulariaceae,Gomortegaceae, Goodeniaceae (goodenia), Greyiaceae, Grossulariaceae(currant), Grubbiaceae, Gunneraceae (gunnera), Gyrostemonaceae,Haloragaceae (water milfoil), Hamamelidaceae (witch hazel),Hernandiaceae (hernandia), Himantandraceae, Hippocastanaceae (horsechestnut), Hippocrateaceae (hippocratea), Hippuridaceae (mare's tail),Hoplestigmataceae, Huaceae, Hugoniaceae, Humiriaceae, Hydnoraceae,Hydrangeaceae (hydrangea), Hydrophyllaceae (waterleaf),Hydrostachyaceae, Icacinaceae (icacina), Idiospermaceae, Illiciaceae(star anise), Ixonanthaceae, Juglandaceae (walnut), Julianiaceae,Krameriaceae (krameria), Lacistemataceae, Lamiaceae (mint, alsoLabiatae), Lardizabalaceae (lardizabala), Lauraceae (laurel),Lecythidaceae (brazil nut), Leeaceae, Leitneriaceae (corkwood),Lennoaceae (lennoa), Lentibulariaceae (bladderwort), Limnanthaceae(meadow foam), Linaceae (flax), Lissocarpaceae, Loasaceae (loasa),Loganiaceae (logania), Loranthaceae (showy mistletoe), Lythraceae(loosestrife), Magnoliaceae (magnolia), Malesherbiaceae, Malpighiaceae(barbados cherry), Malvaceae (mallow), Marcgraviaceae (shingle plant),Medusagynaceae, Medusandraceae, Melastomataceae (melastome), Meliaceae(mahogany), Melianthaceae, Mendonciaceae, Menispermaceae (moonseed),Menyanthaceae (buckbean), Mimosaceae, Misodendraceae, Mitrastemonaceae,Molluginaceae (carpetweed), Monimiaceae (monimia), Monotropaceae (Indianpipe), Moraceae (mulberry), Moringaceae (horseradish tree), Myoporaceae(myoporum), Myricaceae (bayberry), Myristicaceae (nutmeg),Myrothamnaceae, Myrsinaceae (myrsine), Myrtaceae (myrtle), Nelumbonaceae(lotus lily), Nepenthaceae (East Indian pitcherplant), Neuradaceae,Nolanaceae, Nothofagaceae, Nyctaginaceae (four-o'clock), Nymphaeaceae(water lily), Nyssaceae (sour gum), Ochnaceae (ochna), Olacaceae (olax),Oleaceae (olive), Oliniaceae, Onagraceae (evening primrose),Oncothecaceae, Opiliaceae, Orobanchaceae (broom rape), Oxalidaceae (woodsorrel), Paeoniaceae (peony), Pandaceae, Papaveraceae (poppy),Papilionaceae, Paracryphiaceae, Passifloraceae (passionflower),Pedaliaceae (sesame), Pellicieraceae, Penaeaceae, Pentaphragmataceae,Pentaphylacaceae, Peridiscaceae, Physenaceae, Phytolaccaceae (pokeweed),Piperaceae (pepper), Pittosporaceae (pittosporum), Plantaginaceae(plantain), Platanaceae (plane tree), Plumbaginaceae (leadwort),Podostemaceae (river weed), Polemoniaceae (phlox), Polygalaceae(milkwort), Polygonaceae (buckwheat), Portulacaceae (purslane),Primulaceae (primrose), Proteaceae (protea), Punicaceae (pomegranate),Pyrolaceae (shinleaf), Quiinaceae, Rafflesiaceae (rafflesia),Ranunculaceae (buttercup orranunculus), Resedaceae (mignonette),Retziaceae, Rhabdodendraceae, Rhamnaceae (buckthorn), Rhizophoraceae(red mangrove), Rhoipteleaceae, Rhynchocalycaceae, Rosaceae (rose),Rubiaceae (madder), Rutaceae (rue), Sabiaceae (sabia), Saccifoliaceae,Salicaceae (willow), Salvadoraceae, Santalaceae (sandalwood),Sapindaceae (soapberry), Sapotaceae (sapodilla), Sarcolaenaceae,Sargentodoxaceae, Sarraceniaceae (pitcher plant), Saururaceae (lizard'stail), Saxifragaceae (saxifrage), Schisandraceae (schisandra),Scrophulariaceae (figwort), Scyphostegiaceae, Scytopetalaceae,Simaroubaceae (quassia), Simmondsiaceae (jojoba), Solanaceae (potato),Sonneratiaceae (sonneratia), Sphaerosepalaceae, Sphenocleaceae(spenoclea), Stackhousiaceae (stackhousia), Stachyuraceae, Staphyleaceae(bladdernut), Sterculiaceae (cacao), Stylidiaceae, Styracaceae (storax),Surianaceae (suriana), Symplocaceae (sweetleaf), Tamaricaceae (tamarix),Tepuianthaceae, Tetracentraceae, Tetrameristaceae, Theaceae (tea),Theligonaceae, Theophrastaceae (theophrasta), Thymelaeaceae (mezereum),Ticodendraceae, Tiliaceae (linden), Tovariaceae, Trapaceae (waterchestnut), Tremandraceae, Trigoniaceae, Trimeniaceae, Trochodendraceae,Tropaeolaceae (nasturtium), Turneraceae (turnera), Ulmaceae (elm),Urticaceae (nettle), Valerianaceae (valerian), Verbenaceae (verbena),Violaceae (violet), Viscaceae (Christmas mistletoe), Vitaceae (grape),Vochysiaceae, Winteraceae (wintera), Xanthophyllaceae, andZygophyllaceae (creosote bush).

Where the plant is a monocotyledon, the monocotyledon can be selectedfrom the group consisting of corn, wheat, oat, rice, barley, millet,banana, onion, garlic, asparagus, ryegrass, millet, fonio, raishan, nipagrass, turmeric, saffron, galangal, chive, cardamom, date palm,pineapple, shallot, leek, scallion, water chestnut, ramp, Job's tears,bamboo, ragi, spotless watermeal, arrowleaf elephant ear, Tahitianspinach, abaca, areca, bajra, betel nut, broom millet, broom sorghum,citronella, coconut, cocoyam, maize, dasheen, durra, durum wheat, edo,fique, formio, ginger, orchard grass, esparto grass, Sudan grass, guineacorn, Manila hemp, henequen, hybrid maize, jowar, lemon grass, maguey,bulrush millet, finger millet, foxtail millet, Japanese millet, prosomillet, New Zealand flax, oats, oil palm, palm palmyra, sago palm,redtop, sisal, sorghum, spelt wheat, sweet corn, sweet sorghum, taro,teff, timothy grass, triticale, vanilla, wheat, and yam.

Alternatively, the monocotyledon can be from a family selected from thegroup consisting of Acoraceae (calamus), Agavaceae (century plant),Alismataceae (water plantain), Aloeaceae (aloe), Aponogetonaceae (capepondweed), Araceae (arum), Arecaceae (palm), Bromeliaceae (bromeliad),Burmanniaceae (burmannia), Butomaceae (flowering rush), Cannaceae(canna), Centrolepidaceae, Commelinaceae (spiderwort), Corsiaceae,Costaceae (costus), Cyanastraceae, Cyclanthaceae (Panama hat),Cymodoceaceae (manatee grass), Cyperaceae (sedge), Dioscoreaceae (yam),Eriocaulaceae (pipewort), Flagellariaceae, Geosiridaceae, Haemodoraceae(bloodwort), Hanguanaceae (hanguana), Heliconiaceae (heliconia),Hydatellaceae, Hydrocharitaceae (tape grass), Iridaceae (iris),Joinvilleaceae (joinvillea), Juncaceae (rush), Juncaginaceae (arrowgrass), Lemnaceae (duckweed), Liliaceae (lily), Limnocharitaceae (waterpoppy), Lowiaceae, Marantaceae (prayer plant), Mayacaceae (mayaca),Musaceae (banana), Najadaceae (water nymph), Orchidaceae (orchid),Pandanaceae (screw pine), Petrosaviaceae, Philydraceae (philydraceae),Poaceae (grass), Pontederiaceae (water hyacinth), Posidoniaceae(posidonia), Potamogetonaceae (pondweed), Rapateaceae, Restionaceae,Ruppiaceae (ditch grass), Scheuchzeriaceae (scheuchzeria), Smilacaceae(catbrier), Sparganiaceae (bur reed), Stemonaceae (stemona),Strelitziaceae, Taccaceae (tacca), Thurniaceae, Triuridaceae, Typhaceae(cattail), Velloziaceae, Xanthorrhoeaceae, Xyridaceae (yellow-eyedgrass), Zannichelliaceae (horned pondweed), Zingiberaceae (ginger), andZosteraceae (eelgrass).

Where the plant is a gymnosperm, the gymnosperm can be from a familyselected from the group consisting of Araucariaceae, Boweniaceae,Cephalotaxaceae, Cupressaceae, Cycadaceae, Ephedraceae, Ginkgoaceae,Gnetaceae, Pinaceae, Podocarpaceae, Taxaceae, Taxodiaceae,Welwitschiaceae, and Zamiaceae.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention.

Example 1. Use of a Recombinant Bacillus cereus Family Member Displayinga Lipase or an Endoglucanase to Stimulate Plant Growth in Soybeans

The Bacillus subtilis lipase and endoglucanase genes were amplified viapolymerase chain reaction (PCR) using the following primers shown belowin Table 3:

TABLE 3 lipase endoglucanase forward ggatccatggctgaaggatccatgaaacggtcaatc cacaatcc (SEQ ID NO: 39) (SEQ ID NO: 37) reverseggatccttaattcg ggatccttactaatttggttc tattctggcc tgt (SEQ ID NO: 38)(SEQ ID NO: 40)

To create fusion constructs, genes were fused to the native bclApromoter of Bacillus thuringiensis DNA encoding the first 35 amino acidsof BclA (amino acids 1-35 of SEQ ID NO:1) using the splicing byoverlapping extension (SOE) technique. Correct amplicons were clonedinto the E. coli/Bacillus shuttle vector pHP13, and correct clonesscreened by DNA sequencing. Correct clones were electroporated intoBacillus thuringiensis (Cry-, plasmid-) and screened for chloramphenicolresistance. Correct transformants were grown in brain heart infusionbroth overnight at 30° C., plated onto nutrient agar plates, andincubated at 30° C. for 3 days. Spores expressing the fusion construct(BEMD spores) were collected off of the plates by washing in phosphatebuffered saline (PBS) and purified by centrifugation and additionalwashes in PBS. Non-transformed control Bacillus thuringiensis (B.t.)spores were created identically.

Soybeans (strain Jake 011-28-04) were planted 2.54 cm deep in 10 cm deeppots filled with standard loam topsoil. Spores were diluted to aconcentration of 1×10⁴/ml in 50 ml of water and applied to each seed atplanting. Plants were grown under ideal light using T5 lamps, 54 watts,and exposed to 11 hours of light a day under controlled temperatureconditions between 15.5-25.5° C. Plants were watered to saturation everythree days over a two week trial. At the end of two weeks, the height ofeach plant was measured and measurements were normalized to controlBacillus thuringiensis spores. Two independent trials were performed.

Results are shown in Table 4, together with the standard error of themean. In both trials, soybeans grown in the presence of BEMD sporesdisplaying either lipase or endoglucanase grew significantly taller thancontrol B.t. spore treated soybeans (statistical analysis assayed via at-test).

TABLE 4 Soybeans Avg. Height, Comparison to Treatment cm Control SEMTrial #1 Control Bt 14.034 100.0% .521 Lipase, BEMD 17.93 127.8% .395Endocellulase, BEMD 16.31 116.2% .411 Trial #2 Control Bt 15.39 100.0%.749 Lipase, BEMD 19.15 124.4% .428 Endocellulase, BEMD 17.65 114.7%.313

Example 2. Use of a Recombinant Bacillus cereus Family Member Displayingan Endoglucanase to Stimulate Plant Growth in Corn

BEMD spores expressing endoglucanase were created in an identicalfashion as described above in Example 1. Field corn was planted 3.8 cmdeep in 10 cm deep pots filled with standard loam topsoil. Spores,control and BEMD expressing endoglucanase, were diluted to aconcentration of 1×10⁴/ml in 50 ml of water and applied to each plant atplanting. A water-only control was also included. Plants were grownunder ideal light using T5 lamps, 54 watts, and exposed to 11 hours oflight a day under controlled temperature conditions between 15.5-25.5°C. Plants were watered to saturation every three days over the one weektrial. At the end of one week, the height of each plant was measured,and measurements were normalized to control Bacillus thuringiensisspores.

Results are shown in Table 5, together with the standard error of themean. Corn grown in the presence of BEMD spores displaying endoglucanasegrew significantly taller than both control B.t. spore treated soybeansand water-only control plants (statistical analysis assayed via at-test).

TABLE 5 Height, cm Comparison SEM H₂O 15.44   100% 0.318 Bt 18.92122.50% 0.645 BEMD Endo 22.71 143.40% 0.616

Example 3. Use of a Recombinant Bacillus cereus Family Member Displayingan Endoglucanase or a Protease to Stimulate Plant Growth in Wheat

BEMD spores expressing endoglucanase were created in an identicalfashion as described above in Example 1. BEMD spores expressing E. coliprotease PtrB were created using similar methods to those describedabove in Example 1 and the following primers: ggatccatgctaccaaaagcc(forward, SEQ ID NO: 41) and ggatccttagtccgcaggcgtagc (reverse, SEQ IDNO: 42).

Winter hard wheat was planted 2.54 cm deep in 10 cm deep pots filledwith standard loam topsoil. Spores, control and BEMD expressingendoglucanase or protease, were diluted to a concentration of 1×10⁴/mlin 50 ml of water and applied to each plant at planting. A water-onlycontrol was also included. Plants were grown under ideal light using T5lamps, 54 watts, and exposed to 11 hours of light a day under controlledtemperature conditions between 15.5-25.5° C. Plants were watered tosaturation every three days over the one week trial. At the end of oneweek, the height of each plant was measured, and measurements werenormalized to control water only plants.

Results are shown in Table 6, together with the standard error of themean. Wheat grown in the presence of BEMD spores displayingendoglucanase or protease grew significantly taller than control B.t.spore treated or water control soybeans (statistical analysis assayedvia a t-test).

TABLE 6 Height, cm Comparison SEM H₂O 18.11   100% 0.721 Bt Control19.96 110.33% 0.752 BEMD Endo 24.76 136.80% 0.21 BEMD 22.35 123.40%0.354 Protease

Example 4. Use of Recombinant Bacillus cereus Family Members Displayingan Endoglucanase to Stimulate Plant Growth in Ryegrass

BEMD spores expressing endoglucanase were created in an identicalfashion as described above in Example 1. Perennial ryegrass was planted6.4 mm deep in 10 cm deep pots filled with standard loam topsoil.Spores, both control and BEMD expressing endoglucanase, were diluted toa concentration of 1×10⁴/ml in 50 ml of water and applied to each plantat planting. A water-only control was also included. Plants were grownunder ideal light using T5 lamps, 54 watts, and exposed to 11 hours oflight a day under controlled temperature conditions between 15.5-25.5°C. Plants were watered to saturation every three days over the two weektrial. At the end of two weeks, the height of each plant was measured,and measurements were normalized to control water only plants.

Results are shown in Table 7, together with the standard error of themean. Ryegrass grown in the presence of BEMD spores displayingendocellulase grew significantly taller than control B.t. spore treatedor water control ryegrass (statistical analysis assayed via a t-test).

TABLE 7 Height, cm Comparison SEM H₂O 11.43 100.0% 0.137 Bt Control12.29 107.7% 0.128 BEMD 12.78 111.9% 0.137 Endo

Example 5. Use of Recombinant Bacillus cereus Family Members DisplayingEnzymes Involved in the Synthesis or Activation of Plant Hormones toStimulate Plant Growth

The BEMD system can also be used to display enzymes involved in thesynthesis of plant hormones. For example, the plant hormoneindole-3-acetic acid is a potent growth stimulator in plants.Indole-3-acetic acid is synthesized in vivo from tryptophan by theenzymes tryptophan monooxygenase and indole-3-acetamide hydrolase.Indole-3-acetic acid and other auxin hormones can also be synthesized invivo from tryptophan and/or indole by the enzymes nitrilase, tryptophanaminotransferase, indole-3-acetaldehyde dehydrogenase, indole-3-pyruvatedecarboxylase, amine oxidase, tryptophan decarboxylase, and tryptophanside chain oxidases.

The BEMD system can also be used to display enzymes involved in themodification of plant growth hormones into bioactive or inactive forms.For example, nitrilase can be expressed on the BEMD system to catalyzethe conversion of indole-3-acetonitrile into the bioactiveindole-3-acetic acid. Additionally, inactive forms of plant hormones,such as indole-3-acetonitrile can be added into the plant growth mediawith the BEMD-expressed nitrilase to provide a gradual release of activehormone into the plant growth media. Many other inactive or less activeforms of plant hormones can be modified using their correspondingenzymes.

Related plant growth hormones (auxins) include indole-3-pyruvic acid,indole-3-acetaldoxime, indole-3-acetamide, indole-3-acetonitrile,indole-3-ethanol, indole-3-pyruvate, indole-3-butyric acid, phenylaceticacids, 4-chloroindole-3-acetic acid, and indole-3-acetaldoxime. Thesehormones are synthesized from tryptophan and/or indole in vivo via theenzymes tryptophan monooxygenase, indole-3-acetamide hydrolase,nitrilase, nitrile hydrolase, acetolactate synthetase, alphaacetolactate decarboxylase, tryptophan aminotransferase,indole-3-acetaldehyde dehydrogenase, indole-3-pyruvate decarboxylase,amine oxidase, tryptophan decarboxylase, and tryptophan side chainoxidases.

Growth hormones of the cytokinin family can also be synthesized byenzymes expressed in the BEMD system. Examples of cytokinins includekinetin, zeatin (cis and trans), 6-benzylaminopurine, dihydroxyzeatin,N6-(D2-isopentenyl) adenine, ribosylzeatin, N6-(D2-isopentenyl)adenosine, 2 methylthio-cis-ribosylzeatin, cis ribosylzeatin,ribosylzeatin-5-monosphosphate, N6-methylaminopurine,N6-dimethylaminopurine, 2′-deoxyzeatin riboside,4-hydroxy-3-methyl-trans-2-butenylaminopurine, ortho-topolin,meta-topolin, benzyladenine, ortho-methyltopolin, andmeta-methyltopolin. These plant growth stimulating compounds aresynthesized in vivo from mevalonate or adenosine mono/di/triphosphate byenzymes including adenosine phosphate isopentenyltransferases,phosphatases, adenosine kinases, adenine phosphoribosyltransferase,CYP735A, 5′ ribonucleotide phosphohydrolase, adenosine nucleosidases,zeatin cis-trans isomerase, zeatin O-glucosyltransferases,β-glucosidases, cis-hydroxylases, CK cis-hydroxylases, CKN-glucosyltransferases, 2,5-ribonucleotide phosphohydrolases, adenosinenucleosidases, purine nucleoside phosphorylases, and zeatin reductases.

Using methods similar to those described above in Example 1, any ofthese enzymes can be incorporated into the BEMD system for display onBEMD spores by creating a fusion construct comprising the enzyme and atargeting sequence that targets the expressed enzyme to the exosporiumwhen the fusion construct is expressed in a Bacillus cereus familymember. A recombinant Bacillus cereus family member expressing such aconstruct can then be added to the soil or other plant growth medium orapplied directly to plant foliage using methods similar to thosedescribed above in Example 1 for stimulation of plant growth.

The plant growth medium can be supplemented with precursors orsubstrates for the enzymes. For example, the plant growth medium can besupplemented with tryptophan, adenosine monophosphates, adenosinediphosphates, adenosine triphosphates, or indole. Suitableconcentrations of these substrates are between 100 nM and 100 μM.

Example 6. Use of Recombinant Bacillus cereus Family Members DisplayingProteases or Peptidases that Cleave Proteins, Peptides, Proproteins, orPreproproteins into Bioactive Peptides for Stimulation of Plant Growth

Proteases and peptidases can be expressed in the BEMD system that canenzymatically cleave available proteins in the plant growth media tobioactive peptides that can act on the plant directly or indirectly.Examples include the enzymatic cleavage of soybean meal, yeast extract,or other protein rich meals added to the plant growth medium into activepeptides that can directly stimulate plant growth. Bioactive peptidesgenerated by enzymatic cleavage of protein meals include RHPP and RKN16D10, potent stimulators of plant root development. Additionally,proproteins or preproproteins can be cleaved into active forms byBEMD-expressed proteases and peptidases to their bioactive forms.Inactive proproteins or preproproteins can be added in the plant growthmedium to facilitate their gradual cleavage by BEMD proteases and slowrelease of bioactive proteins.

Using methods similar to those described above in Example 1, any ofthese proteases and peptidases can be incorporated into the BEMD systemfor display on BEMD spores by creating a fusion construct comprising theprotease or peptidase and a targeting sequence that targets theexpressed enzyme to the exosporium when the fusion construct isexpressed in a Bacillus cereus family member. A recombinant Bacilluscereus family member expressing such a construct can then be added tosoil or other plant growth medium supplemented with soybean meal, yeastextract, or another-protein-rich meal for stimulation of plant growth.The soybean meal, yeast extract, or other protein-rich meal is suitablyadded to the plant growth medium in the form of a liquid compositioncomprising about 10 μg/L to about 100 mg/L of the protein meal, yeastextract, or other protein-rich meal.

Example 7. Use of BEMD Spores Expressing the Protease PtrB forStimulation of Plant Growth

BEMD spores expressing E. coli protease PtrB were created as describedabove in Example 3. Soybean seeds were planted 2.54 cm deep in 10 cmdeep pots filled with standard loam topsoil. Spores, both control andBEMD expressing protease, were diluted to a concentration of 1×10⁴/ml in50 ml of water and applied to each plant at planting. A water-onlycontrol was also included. Soybean meal at 25 mg/pot was added in waterat planting. Plants were grown under ideal light using T5 lamps, 54watts, and exposed to 13 hours of light a day under controlledtemperature conditions between 15.5-25.5° C. Plants were watered tosaturation every three days over the one week trial. At the end of twoweeks, the height of each plant was measured, and measurements werenormalized to control water only plants.

Results are shown in Table 8, together with the standard error of themean as a percentage of water control. Soy grown in the presence of BEMDspores displaying protease grew significantly taller than control B.t.spore treated or water control soybeans (statistical analysis assayedvia a t-test). The addition of soybean meal to water control or B.thuringiensis control plants had little effect. By contrast, in thepresence of the soybean meal and the BEMD protease system, the soybeanplants responded significantly over all other treatments.

TABLE 8 SEM, as Height Normalized percentage Treatment Soybean Meal (cm)to water of water Water only No 12.10   100% 3.1% Water only 25 mg/pot12.43 102.7% 7.4% B. thuringiensis No 12.52 103.5% 5.2% B. thuringiensis25 mg/pot 11.99  99.1% 5.0% BEMD Protease No 12.97 107.2% 6.1% BEMDProtease 25 mg/pot 14.44 119.3% 4.8%

Example 8. Use of Recombinant Bacillus cereus Family Members DisplayingProteins or Peptides Involved in the Stimulation of Plant Growth

The BEMD system can also be used to display proteins or peptides thatare directly involved in the promotion of plant growth. For example,plant peptide hormones or non-hormone peptides that stimulate plantgrowth can be expressed in the BEMD system. For example, non-hormonepeptides that directly bind to and active plant receptors can beexpressed in the BEMD system to directly act on receptors in the plantand roots of target plants. Such peptide hormones and non-hormonepeptides include phytosulfokine, calcalva 3 (CLV3), systemin, RKN 16D10,Hg-Syv46, eNOD40, NOD family proteins, ZmlGF, SCR/SP11 family proteinsand peptides, RHPP, POLARIS, and KTI. These peptides and relatedpeptides can be expressed in the BEMD system and delivered to plantgrowth medium or directly applied to foliage to stimulate plant growth.

Using methods similar to those described above in Example 1, any ofthese proteins or peptides can be incorporated into the BEMD system fordisplay on BEMD spores by creating a fusion construct comprising theenzyme and a targeting sequence that targets the expressed enzyme to theexosporium when the fusion construct is expressed in a Bacillus cereusfamily member. A recombinant Bacillus cereus family member expressingsuch a construct can then be added to the soil or other plant growthmedium or applied directly to plant foliage using methods similar tothose described above in Example 1 for stimulation of plant growth.

Example 9. Use of BEMD Spores Expressing POLARIS or KTI for Stimulationof Plant Growth

BEMD spores expressing the plant peptide POLARIS and soy peptide KTIwere created by synthesizing genes coding for the POLARIS or KITpeptides linked to the targeting sequence of SEQ ID NO: 60. The geneswere then introduced genes into Bacillus thuringiensis and spores weremade as described in Example 1. Soybean seeds were planted 2.54 cm deepin 10 cm deep pots filled with standard loam topsoil. BEMD sporesexpressing POLARIS or KTI were diluted to a concentration of 1×10⁴/ml in50 ml of water and applied to each plant at planting. A water-onlycontrol was also included. Pure POLARIS and KTI peptides were alsotested for their effects on soybeans at 0.05 mg/pot. Plants were grownunder ideal light using T5 lamps, 54 watts, and exposed to 13 hours oflight a day under controlled temperature conditions between 15.5-25.5°C. Plants were watered to saturation every three days over the two weektrial. At the end of two weeks, the height of each plant was measured,the roots measured, and measurements were normalized to control wateronly plants.

Results are shown in Table 9, together with the standard error of themean as a percentage of water control. Soy grown in the presence of BEMDspores displaying POLARIS grew taller and had a slight increase in rootdevelopment than water control soybeans. The presence of free KTIpeptide led to a significant stunting of the plants, losing between 6-8%of their heights, but adding 15% to the length of the roots. Expressionof KTI on the BEMD system led to the root growth benefit, but withoutthe stunting effect on the plant height. Importantly, the presence ofthe Bacillus thuringiensis control spores with the free KTI peptide didnot prevent the stunting effect of KTI, while the BEMD with KTIdisplayed no such stunting.

TABLE 9 Roots Height, Normalized Normalized Treatment Peptide to WaterSEM to Water SEM Water No 100% 6.8%  100% 4.3% Water KTI, 115% 8.4%91.8% 3.1% 0.05 mg/Pot BEMD No 106.3%   7.9% 107.3%  1.7% POLARIS BEMDKTI No 113.3%   5.8% 99.4% 3.4% B. KTI, 115% 7.7% 93.4% 4.2%thuringiensis 0.05 mg/pot

Example 10. Use of Recombinant Bacillus cereus Family Members DisplayingEnzymes that Degrade or Modify a Bacterial, Fungal, or Plant NutrientSource to Stimulate Plant Growth and/or Process Nutrients

The BEMD system can also be used to display enzymes that degrade ormodify beneficially a bacterial, fungal, or plant nutrient sourcepresent in soil or another plant growth medium. Such enzymes degradeproducts present in the soil or other plant growth medium into formsthat can easily be taken up by plants and/or the beneficial bacteriaand/or fungi of the rhizosphere. Such enzymes include, for example,glucoside hydrolases to degrade complex carbohydrates, cellulases todegrade cellulose; lipases to degrade lipids, including oil, fats, andwaxes; lignin oxidases to degrade lignin and humic acids; proteases todegrade polypeptides; phospholipases to degrade membranes; amidases andnitrogenases to recover nitrogen; amylases to process starches;nucleases to recover nucleotides, pectinases to break down pectin,sulfatases to recover sulfur, and xylanases to break down xylans andarabinoxylans. The resultant products, including simple sugars, aminoacids, fatty acids, and other nutrients will be readily available fordirect uptake by plants and/or for stimulating beneficial bacteriaand/or fungi to grow and thrive in the rhizospheres of the plants.

In addition, enzymes and other biological molecules can be utilized torelease or sequester phosphate, nitrogen, and other key elementalnutrients for plant uptake from their various organic and inorganicforms in soil. For example, phosphatases can be used to degradephosphates in the environment into usable inorganic phosphates for plantuse. The phosphates can be naturally occurring phosphates present in aplant growth medium. Alternatively or in addition, the plant growthmedium can be supplemented with phosphates such as trimetaphosphate, acommon agricultural amendment. Examples of useful phosphatases includephosphoric monoester hydrolases, phosphomonoesterases, phosphoricdiester hydrolases, phosphodiesterases, triphosphoric monoesterhydrolases, phosphoryl anhydride hydrolases, pyrophosphatases, phytase,trimetaphosphatases, and triphosphatases. For example, the enzymestrimetaphosphatase, triphosphatase, and pyrophosphatase sequentiallybreak down trimetaphosphate into usable inorganic phosphate.

The nitrogenase family of enzymes converts atmospheric nitrogen (N2)into ammonia, thereby converting nitrogen that would otherwise beinaccessible to plants into a usable form. Suitable enzymes belong tothe Nif family of nitrogenases.

Chemical energy can also be directly added into the plant growth mediumas adenosine-3-triphosphate, ferrodoxin, or additional enzymes thatcreate such energy into the BEMD system. These are cofactors for thenitrogenases and are limited in soil. Thus, such cofactors can be addedto soil to enhance the reactions described above.

Other supplements that can be added to the plant growth medium includestarches, cellulose and cellulose derivatives, pectins, xylans andarabinoxylans, fats, waxes, oils, phytic acids, lignins, humic acids,and other nutrient sources that the above enzyme classes exert activityupon.

Using methods similar to those described above in Example 1, any ofthese enzymes can be incorporated into the BEMD system for display onBEMD spores by creating a fusion construct comprising the enzyme and atargeting sequence for targeting the fusion construct to the exosporiumof a Bacillus cereus family member. The fusion construct can then beexpressed in a Bacillus cereus family member, and this recombinantBacillus cereus family member can be added to soil or another plantgrowth medium using methods similar to those described above in Example1 for stimulation of plant growth.

Example 11. Use of BEMD Spores Expressing a Phosphatase for Stimulationof Plant Growth

BEMD spores expressing Bacillus subtilis Phosphatase A4 (PhoA4) werecreated by synthesizing a gene coding for PhoA4 linked to the targetingsequence of SEQ ID NO: 60. This gene was then introduced into Bacillusthuringiensis and spores were made as in Example 1. Corn was planted2.54 cm deep in 10 cm deep pots filled with standard loam topsoil. BEMDspores expressing PhoA4, were diluted to a concentration of 1×10⁴/ml in50 ml of water and applied to each plant at planting. A water-onlycontrol was also included. Polyphosphate was added to pots in liquid ata rate of 0.5 mg/pot. Plants were grown under ideal light using T5lamps, 54 watts, and exposed to 13 hours of light a day under controlledtemperature conditions between 15.5-25.5° C. Plants were watered tosaturation every three days over the two week trial. At the end of twoweeks, the height of each plant was measured, and measurements werenormalized to control water only plants.

Results are shown in Table 10. Corn grown in the presence of BEMD sporesdisplaying PhoA4 exhibit enhanced growth, especially in the presence ofadded polyphosphate. This effect was greater than the effect of thepolyphosphate alone.

TABLE 10 Growth, Treatment Additive Comparison to Water Water None  100% Water Polyphosphate 110.8% BEMD PhoA4 None 108.3% BEMD PhoA4Polyphosphate 114.8%

Example 12. Use of Recombinant Bacillus cereus Family Members DisplayingEnzymes Involved in the Synthesis of 2,3-Butanediol or the Synthesis orActivation of Gibberellic Acid for Stimulation of Plant Growth

The BEMD system can also be used display enzymes involved in thesynthesis of the plant-growth promoting compound 2,3-butanediol. Invivo, 2,3-butanediol is synthesized by beneficial bacteria and fungi inthe rhizosphere from acetoin, diacetyl, acetolactate, or pyruvate by theenzymes acetolactate synthetase, α-acetolactate decarboxylase, pyruvatedecarboxylase, diacetyl reductase, butanediol dehydrogenases, andacetoin reductase.

The BEMD system can also be used to display enzymes involved in thesynthesis or activation of the plant-growth promoting compoundgibberellic acid. Gibberellic acid can be produced from inactive or lessactive forms via the action of enzymes, including but not limited tohydroxylamine reductases, 2-oxogluturate dioxygenases, gibberellin 2B/3Bhydrolases, gibberellin 3-oxidases, and gibberellin 20-oxidases.

Any of these enzymes can be incorporated into the BEMD system fordisplay on BEMD spores using methods similar to those described above inExample 1. A fusion construct can be prepared that comprises the enzymeand a targeting sequence that targets the enzyme to the exosporium whenthe fusion construct is expressed in a Bacillus cereus family member.The fusion construct is then expressed in a Bacillus cereus familymember, and the Bacillus cereus family member is added to soil oranother plant growth medium for stimulation of plant growth.

To increase the effect of the enzymes displayed on BEMD, the soil can besupplemented with substrates for the enzymes. For example, the soil orother plant growth medium can be supplemented with acetoin, which is asubstrate for acetoin reductase; pyruvate, which is a substrate forpyruvate decarboxylase; diacetyl, which is a substrate for diacetylreductase; and/or acetolactate, which is a substrate for acetolactatedecarboxylase. Alternatively or in addition, the soil or other plantgrowth medium can be supplemented with less potent or inactive forms ofgibberellic acid, which will converted into more active forms by theenzymes described above in the soil or other plant growth medium.

Example 13. Use of Recombinant Bacillus cereus Family Members DisplayingProteases for Protecting Plants from Pathogens

The BEMD system can also be used display proteases that protect plantsfrom one or more pathogens. For example, certain bacterial pathogens cancommunicate between individual members via secretion of bacteriallactone homoserines or related signaling molecules. Thus, proteasesspecific for bacterial lactone homoserine signaling molecules canprotect plants from such bacterial pathogens by disrupting communicationbetween bacteria, a step essential for the bacteria to secrete toxinsand upregulate virulence factors. Suitable proteases specific forbacterial lactone homoserine signaling molecules include endopeptidasesand exopeptidases.

Proteases specific for bacterial lactone homoserine signaling moleculescan be incorporated into the BEMD system using methods similar to thosedescribed above in Example 1. A fusion construct can be prepared thatcomprises the protease and a targeting sequence that targets theprotease to the exosporium when the fusion construct is expressed in aBacillus cereus family member. The fusion construct is then expressed ina Bacillus cereus family member, and the Bacillus cereus family memberis added to soil or another plant growth medium. The protease can thendegrade the bacterial lactone homoserine signaling molecules, blocking akey step in the virulence of these organisms and thereby helping toprotect the plant from these pathogens. Other proteases and peptidaseswork effectively in this capacity on the BEMD system as demonstratedabove in Example 6 and 7.

Example 14. Use of Recombinant Bacillus cereus Family Members DisplayingAntimicrobial Proteins and Peptides for Protecting Plants from Pathogens

The BEMD system can also be used display enzymes that exhibitantibacterial and/or antifungal activities that can help protect plantsfrom one or more pathogens. For example, antimicrobial proteins andpeptides such as bacteriocins, lysozymes (e.g., LysM), siderophores,conalbumin, albumin, lactoferrins (e.g., LfcinB), or TasA can all beexpressed in the BEMD system to exert their effect on bacterial andfungal pathogens of plants. Bacteriocins, albumin, conalbumin,lysozymes, and lactoferrin exert direct antimicrobial action on theirtargets, whereas siderophores bind essential nutrients that pathogensrequire for virulence. For example, the peptide LfcinB of lactoferrin,when expressed on the surface of the BEMD system would lyse bacteriacells that are susceptible to the lactoferrin peptides in the plantgrowth medium. These proteins and peptides have specific action onselect microbes, and can selectively target a group of pathogens withoutobstructing all microbes in the plant growth medium.

Any of these proteins or peptides can be incorporated into the BEMDsystem for display on BEMD spores using methods similar to thosedescribed above in Example 1. A fusion construct can be prepared thatcomprises the enzyme and a targeting sequence that targets the enzyme tothe exosporium when the fusion construct is expressed in a Bacilluscereus family member. The fusion construct is then expressed in aBacillus cereus family member, and the Bacillus cereus family member isadded to soil or another plant growth medium for protection of plantsfrom one or more pathogens.

Example 15. Use of BEMD Spores Expressing Antimicrobial Peptides forProtecting Plants from Bacteria

Genes were synthesized that coded for either of two antimicrobialpeptides, LfcinB (derived from bovine lactoferrin) and LysM (derivedfrom chicken lysozyme), linked to a BclA targeting sequence (SEQ ID NO:60), under the control of the BclA promoter (SEQ ID NO: 85). The geneswere introduced into Bacillus thuringiensis BT013A and spores were madeby growing an overnight culture of the transformed Bacillus in brainheart infusion broth, plating onto nutrient agar plates at 30° C. andallowing to grow for 3 days. Spores were washed off the plates andrinsed 3× in PBS. Staphylococcus epidermidis cultures were grownovernight in TSB broth at 37° C. The overnight culture was thenpelleted, washed in PBS, and resuspended in PBS at an Abs595=0.2.1×10⁴BEMD expressing the LysM or LfcinB peptides was incubated in thePBS with the S. epidermidis for 3 hours at 37° C., with shaking. Acontrol sample of S. epidermidis was left untreated (no BEMD spores).After the 3 hour incubation, dilution plates of the S. epidermidis weremade and incubated at 37° C. overnight. S. epidermidis cultures werecounted the next day, and percent killing quantified. In Table 11 below,a record of the killing activity was recorded. The BEMD expressedpeptides killed a significant number of S. epidermidis cells. This woulddirectly translate into killing of bacteria on the rhizosphere, seed, orother plant material. The selection of peptides specific to certainclasses of bacteria can also skew the population of the microorganismsnear the plant in a beneficial way, or can selectively target keypathogens.

TABLE 11 Treatment Survival % Killed None 100% 0% BEMD LysM 71% 29% BEMDLfcinB 23% 77%

Example 16. Use of Recombinant Bacillus cereus Family Members DisplayingEnzymes for Protecting Plants from Pathogens

The BEMD system can also be used display enzymes that protect plantsfrom one or more pathogens. For example, yeast and mold cell walls aredegraded by enzymes such as β-1,3-glucanases, β-1,4-glucanases,β-1,6-glucanases, chitosinases, chitinases, chitosinase-like proteins,and lyticases. Bacteria cell walls are degraded by enzymes selected fromproteinases, proteases, mutanolysin, stapholysin, and lysozymes. Each ofthese cell wall degrading enzymes can be expressed on the BEMD systemand added to plant growth medium for selective inhibition of pathogenicmicrobes in the rhizosphere.

The BEMD system can also be used to display enzymes or proteins thatprotect plants from insect or worm pathogens, for example by suppressinginsect and/or worm predation of desired plants. Examples of suchproteins and enzymes of interest include endotoxins, Cry toxins, otherinsecticidal protein toxins, protease inhibitors, cysteine proteases,the Cry5B protein, the Cry 21A protein, chitinase, protease inhibitorproteins, protease inhibitor peptides, trypsin inhibitors, and arrowheadprotease inhibitors.

Any of these proteins or peptides can be incorporated into the BEMDsystem for display on BEMD spores using methods similar to thosedescribed above in Example 1. A fusion construct can be prepared thatcomprises the enzyme and a targeting sequence that targets the enzyme tothe exosporium when the fusion construct is expressed in a Bacilluscereus family member. The fusion construct is then expressed in aBacillus cereus family member, and the Bacillus cereus family member isadded to soil or another plant growth medium for protection of plantsfrom pathogens.

Example 17. Use of BEMD Spores Expressing an Antifungal Enzyme forProtecting Plants, and Demonstration of Efficacy Against Saccharomyces

A gene was synthesized that encoded an antifungal enzyme,β-1,3-glucanase from Bacillus subtilis, linked to a BclA targetingsequence (SEQ ID NO: 60) under the control of the BclA promoter (SEQ IDNO: 85). The gene was and introduced into Bacillus thuringiensis BT013Aand pores were made by growing an overnight culture of the transformedBacillus in brain heart infusion broth, plating onto nutrient agarplates at 30° C., and allowing to grow for 3 days. Spores were washedoff the plates and rinsed 3× in PBS. Saccharomyces cerevisiae cultureswere grown overnight in YZ broth at 37° C. The overnight culture wasthen pelleted, washed in PBS, and resuspended in PBS at an Abs595=0.2.1×10⁴ BEMD expressing β-1,3-glucanase was incubated in the PBS with theSaccharomyces for 1 hour at 37° C., with shaking. A control sample ofSaccharomyces was left untreated (no BEMD spores). After the 3 hourincubation, dilution plates of the Saccharomyces were made and incubatedat 37° C. overnight. Saccharomyces cultures were counted the next day,and percent killing quantified. In Table 12 below shows the killingactivity of the BEMD spores expressing β-1,3-glucanase. TheBEMD-expressed enzyme killed a significant number of Saccharomycescells. This would directly translate into killing of fungalmicroorganisms on the rhizosphere, seed, or other plant material. Theselection of proteins specific to certain classes of fungi can also skewthe population of the microorganisms near the plant in a beneficial way,or can selectively target key fungal pathogens.

TABLE 12 Treatment Survival % Killed None 100% 0% BEMD β-1,3-glucanase83% 17%

Example 18. Use of Recombinant Bacillus cereus Family Members DisplayingPlant Immune System Stimulatory Peptides or Proteins for ProtectingPlants from Pathogens

The BEMD system can also be used display plant immune system enhancerpeptides and proteins. These proteins can be expressed on the outside ofthe BEMD spore and delivered into the plant growth medium to stimulatethe plant immune system to allow the plant to protect itself from plantpathogens. Example proteins and peptides include harpin, α-elastins,β-elastins, systemins, phenylalanine ammonia-lyase, elicitins,defensins, cryptogein, and flagellin proteins and peptides. Exposure ofplants to these proteins and peptides will stimulate resistance to manyplant pathogens in plants.

Any of these proteins or peptides can be incorporated into the BEMDsystem for display on BEMD spores using methods similar to thosedescribed above in Example 1. A fusion construct can be prepared thatcomprises the enzyme and a targeting sequence that targets the enzyme tothe exosporium when the fusion construct is expressed in a Bacilluscereus family member. The fusion construct is then expressed in aBacillus cereus family member, and the Bacillus cereus family member isadded to soil or another plant growth medium for protection of plantsfrom pathogens.

Example 19. Use of Recombinant Bacillus cereus Family Members Displayinga Root or Leaf Binding Protein or Peptide to Immobilize the RecombinantBacillus cereus Family Member on a Root System of a Plant or on PlantLeaves

Root and leaf binding proteins and peptides can also be incorporatedinto the BEMD system to allow the BEMD spores to be immobilized on aroot system or on leaves of a plant. Display of such root or leafbinding ligands on the BEMD spores allows for targeting of the spores tothe root system of a plant or to substructures of the root system or tothe leaves or to substructures of leaves to maintain the BEMD spores atan optimal location for other displayed biological molecules and enzymesto be effective.

For example, rhicadhesin is a root binding ligand that binds to roothairs. Thus, display of rhicadhesin on the BEMD spores thus targets thespores to root hairs. Additional proteins that could be utilized forselective binding to plant roots or leaves include adhesins, flagellin,omptins, lectins, pili proteins, curlus proteins, intimins, invasins,agglutinin, afimbrial proteins, TasA, or YuaB.

Such root or leaf binding proteins and peptides can be incorporated intothe BEMD system using methods similar to those described above inExample 1. A fusion construct can be prepared that comprises the root orleaf binding protein or peptide and a targeting sequence that targetsthe protein or peptide to the exosporium when the construct is expressedin a Bacillus cereus family member. The fusion construct containing theroot or leaf binding ligand is then expressed in a Bacillus cereusfamily member. Such fusion constructs can be coexpressed with one ormore additional fusion constructs comprising any of the beneficialenzymes discussed herein (e.g., an enzyme involved in the synthesis of aplant hormone, an enzyme that degrades a nutrient source, or a proteasesthat protects a plant from a pathogen). The recombinant Bacillus cereusfamily member is added to soil or another plant growth medium, orapplied to the leaves of a plant. The root or leaf binding ligandtargets the Bacillus cereus family member to the root system of theplant or to the leaves of the plant and immobilizes it there, thusallowing the coexpressed fusion construct to exert its effects in closeproximity to the root or leaf system.

Example 20. Use of Recombinant Bacillus cereus Family Members DisplayingProteins or Enzymes to Enhance Stress Resistance of Plants

Proteins, peptides, and enzymes that enhance stress resistance in aplant can be incorporated into the BEMD system and delivered to targetplants via addition to roots, leaves, or the plant growth medium. Duringperiods of stress, plants release stress-related compounds, includingaminocyclopropane-1-carboxlic acid (ACC), reactive oxygen species, andothers, resulting in a negative impact on plant growth. The BEMD systemcan be used to display enzymes that degrade such stress-relatedcompounds, such as aminocyclopropane-1-carboxylic acid deaminase,superoxide dismutases, oxidases, catalases, and other enzymes that acton reactive oxygen species. Such enzymes reduce the amount of thesestress-related compounds and allow plants to continue to grow and eventhrive under stressed conditions.

Any of these proteins or peptides can be incorporated into the BEMDsystem for display on BEMD spores using methods similar to thosedescribed above in Example 1. A fusion construct can be prepared thatcomprises the enzyme and a targeting sequence that targets the enzyme tothe exosporium when the fusion construct is expressed in a Bacilluscereus family member. The fusion construct is then expressed in aBacillus cereus family member, and the Bacillus cereus family member isadded to soil or to another plant growth medium or applied to the leavesof a plant for enhancing the stress resistance of a target plant.

Example 21. Preparation of BEMD Spores Expressing the Protective EnzymeCatalase

A gene was synthesized that encoded the protective enzyme catalase fromBacillus cereus linked to a BetA targeting sequence (SEQ ID NO: 61)under the control of the BetA promoter (SEQ ID NO: 86). This gene wasand introduced into Bacillus thuringiensis BT013A. Spores were made bygrowing an overnight culture of the transformed Bacillus and wildtypestrain in brain heart infusion broth, plating onto nutrient agar platesat 30° C., and allowing to grow for 3 days. Spores were washed off theplates and rinsed 3× in PBS. 3 drops of hydrogen peroxide was added toeach spore pellet. The enzyme catalase converts the hydrogen peroxideinto water and 02 gas. The control spores did not bubble, while theBEMD-catalase spores readily did, demonstrating enzyme activity on thesurface of the spores. Other protective enzymes can be displayed in asimilar fashion and delivered to the plant to act upon free radicalsproduced during stress by the plants.

Example 22. Use of Recombinant Bacillus cereus Family Members DisplayingProteins or Enzymes that Protect Seeds or Plants from an EnvironmentalStress

Proteins, peptides, and enzymes that protect a plant from anenvironmental stress can be incorporated into the BEMD system anddelivered to target plants via addition to roots, leaves, fruit, or theplant growth medium. During periods of freezing, plants can be damagedby the effect of ice. The BEMD system can be used to display peptides,proteins, or enzymes that protect plants from such effects. For example,the BEMD system can be used to display choline dehydrogenases, which actby producing protective products that protect the plant or seed fromfrost. Substrates for these enzymes (e.g., choline and/or cholinederivatives) can also be added to the plant growth medium. Addition ofsuch substrates can enhance the amount of protectant (betaine andrelated chemistries) produced in the plant environment by the BEMDexpressed enzymes. Betaine derivatives are known to protect seeds fromcold stress.

Any of these proteins or peptides can be incorporated into the BEMDsystem for display on BEMD spores using methods similar to thosedescribed above in Example 1. A fusion construct can be prepared thatcomprises the enzyme and a targeting sequence that targets the enzyme tothe exosporium when the fusion construct is expressed in a Bacilluscereus family member. The fusion construct is then expressed in aBacillus cereus family member, and the Bacillus cereus family member isadded to soil or to another plant growth medium or applied to the leavesof a plant for protecting the plant from environmental stresses andfactors.

Example 23. Enhanced Expression of Fusion Constructs on the BEMD Systemby Use of Enhanced or Alternative Promoter Elements

The BEMD system can display a wide range of proteins, peptides, andenzymes using one or more of the targeting sequences described herein.Some of these targeting sequences have a high affinity for theexosporium which would be beneficial for fusion protein expression, buttheir low fusion protein expression level limits their use on the BEMDsystem. For such fusion proteins and sequences, alternativehigh-expression sporulation promoters can be used instead of the nativepromoters.

For example, SEQ ID NO: 13 (amino acids 1-39 of B. weihenstephensisKBAB4 gene 3572) provides a very effective N-terminal sequence for thedelivery of proteins to the exosporium of Bacillus cereus familymembers, as shown in Table 13 below. All genes were synthesized in theircomplete form (including promoter regions and regions coding for fusionproteins) as described herein. When the native promoter elements for B.weihenstephensis KBAB4 gene 3572 (SEQ ID NO: 88) were used to express afusion protein comprising the targeting sequence of SEQ ID NO: 13 fusedto a β-galactosidase enzyme (from E. coli), a low level of fusionprotein was expressed, leading to a reduction in enzyme activity on thesurface of the spore. Enzyme activity was measure by the conversion of0.5M o-nitrophenylgalactoside in solution over 10 minutes. Enzymeconversion was measured with a spectrophotometer at ABS₅₄₀. Replacementof the native promoter elements of the B. weihenstephensis KBAB4 gene3572 with the high-expression promoters of SEQ ID NO: 86 (B. anthracisBetA/BAS3290) or SEQ ID NO: 89 (B. weihenstephensis KBAB4 YVTNβ-propeller protein) led to a dramatic increase in the enzymaticactivity of the spores. On the other hand, replacement of the nativepromoter elements for B. weihenstephensis KBAB4 gene 3572 with thepromoter native to B. anthracis Sterne BAS1882 (SEQ ID NO: 87) led to adecrease in the enzymatic activity of the spores. The expression levelof the targeting sequence of SEQ ID NO: 13 fused to β-galactosidase wasmuch lower (0.38×) when driven by the promoter of BAS1882 (SEQ ID NO:87), and was greatly improved when driven from the BetA promoter (SEQ IDNO: 86) or YVTN protein promoter (SEQ ID NO: 89).

TABLE 13 β-galactosidase activity on BEMD Fold Promoter Fusion Proteinsystem, normalized Change SEQ ID NO: 88 SEQ ID NO: 13 - β-   100%galactosidase SEQ ID NO: 86 SEQ ID NO: 13 - β- 213.4% 2.13Xgalactosidase SEQ ID NO: 89 SEQ ID NO: 13 - β- 220.7% 2.21Xgalactosidase SEQ NO: ID 87 SEQ ID NO: 13 - β-  38.1% 0.38Xgalactosidase

Example 24. Isolation and Identification of Plant-Growth PromotingBacterial Strains

Soil samples from rhizospheres of the healthiest and most resistantpotato (Solanum tuberosum), yellow summer squash (Cucurbita pepo),tomato (Solanum lycopersicum), and pole bean (Phaseolus coccineus)plants were collected, diluted in sterile water, and spread ontonutrient agar plates. Bacterial isolates that demonstrated high growthrates and were able to be passaged and propagated were selected forfurther study. The selected strains were grown in minimal media (KH₂PO₄3 g, Na₂HPO₄ 6 g, NH₄Cl 1 g, NaCl 0.50 g, MgSO₄ 7H₂O 0.15 g, CaCl₂) 2H₂O0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30° C.)of selected strains were spun down, media decanted off, and resuspendedin an equal amount of distilled water. Ten lettuce seeds per treatmentwere planted at a depth of 1 cm in loam top soil (Columbia, Mo.) thatwas sieved to remove large debris. Seeds were inoculated at planting in4 cm pots with 0.5 μl of resuspended bacteria in water mixed into 10 mlof H₂O. Ten ml of H₂O was sufficient to deliver the bacteria into the 3in³ (7.62 cm³) of soil as well as saturate the soil for propergermination of seeds. Plants were grown at temperatures between 65-75°F. (18-24° C.) with 11 hours of light/day, and 5 ml of watering every 3days. After one week, plant heights and leaf diameters, as well asoverall health of the plants were collected. Initial screening ofrhizosphere isolates resulted in obtaining greater than 200 distinctspecies of bacteria and fungi from the rhizosphere of the four plants.Some of the bacterial species are described in Table 14. Identifiedstrains are indicated by their proper bacterial identifications. Otherstrains are indicated by their unknown identification number. Inoculantsgiving results near control (+/−2%) were not included in the table.

TABLE 14 Butterhead Lettuce Bacterial Inoculant Avg. Height (cm)Comparison SEM Uninoculated 1.8 Control .07 Paracoccus kondratiavae 2111.1% .05 NC35 B. aryabhattai CAP53 3.65 202.8% .45 B. flexus BT0542.45 136.1% .11 Bacillus mycoides strain 2.17 120.4% .21 BT155 B.aryabhattai CAP56 2.1 116.7% .20 B. nealsonii BOBA57 2.8 155.6% .03 E.cloacae CAP12 2.4 133.3% .41 Unknown 8 1.77 77.8% .65 Unknown 122 1.9105.6% .11 Unknown 15 1.4 77.8% .41 Unknown 39 1.8 100.0% .20 Unknown401 2 111.1% .21 Unknown 402 1.53 85.2% .27 Unknown 41 1.45 80.6% .31Unknown 42 1.4 77.8% .15 Unknown 44 2.2 133.3% .08 Unknown 51 1.83102.9% .21

Bacterial strains that produced the greatest effect on the overall planthealth and plant height in the initial lettuce trial were subjected tofurther identification. Bacterial strains were grown overnight in LuriaBertani broth at 37° C., and overnight cultures were spun down in acentrifuge. Media was decanted and the remaining bacterial pellet wassubjected to chromosomal DNA isolation using the Qiagen BacterialChromosomal DNA Isolation kit. Chromosomal DNA was subjected to PCRamplification of the 16S rRNA coding regions using the primers E338F5′-ACT CCT ACG GGA GGC AGC AGT-3′ (SEQ ID NO: 122), E1099R A 5′-GGG TTGCGC TCG TTG C-3′ (SEQ ID NO: 123), and E1099R B 5′-GGG TTG CGC TCG TTAC-3′ (SEQ ID NO: 124). PCR amplicons were purified using a Promega PCRpurification kit, and the resultant amplicons were diluted and sent tothe University of Missouri DNA Core for DNA sequencing. DNA sequenceswere compared to the NCBI BLAST database of bacterial isolates, andgenus and species were identified by direct comparison to known strains.Top identified species are indicated in Table 14. In many cases, 16SrRNA DNA sequences were only able to delineate the genus of the selectedbacterial strain. In cases where a direct identification was notforthcoming, additional biochemistry analyses, using methods standard inthe field, were performed to differentiate strains at the species andstrain levels, and are listed in Table 15.

TABLE 15 E. cloacae P. kondratiavae B. aryabhattai B. flexus B. mycoidesB. aryabhattai B. nealsoni Test CAP12 NC35 CAP53 BT054 BT155 CAP56BOBA57 Urease − − − − − − + Catalase + + + + + + + Oxidase − + + + − − −Nitrate + + − + + − + Growth, 5% NaCl + − + + − + + Growth, 7.5% NaCl −− + + − + − Growth, 42° C. + + + + + + + Growth, 50° C. − − + + − + −Growth, pH 5 + − + + − + − Growth, pH 9 + + + + + + + Acid, Cellobiose +− + + + + − Acid, Lactose + − + + + − + Acid, Starch − − − + − + −

Example 25. Isolation and Identification of Additional Plant-GrowthPromoting Bacterial Strains

Soil samples from agricultural fields near Gas, Kans. were collected,diluted in sterile water, and spread onto nutrient agar plates.Bacterial isolates that demonstrated high growth rates and were able tobe passaged and propagated were selected for further study. The selectedstrains were grown in minimal media (KH₂PO₄ 3 g, Na₂HPO₄ 6 g, NH₄Cl 1 g,NaCl 0.50 g, MgSO₄ 7H₂O 0.15 g, CaCl₂) 2H₂O 0.013 g, and glucose 1 g,per L dry weight). Overnight cultures (30° C.) of selected strains werespun down, media decanted off, and resuspended in an equal amount ofdistilled water. Corn seeds were coated with commercial seed polymermixed with water alone (1.6 μl per seed total) or commercial seedpolymer containing selected bacterial strains (1.6 μl per seed total).Coated seeds were planted in (3 inch) 7.62 cm diameter pots at a depthof 1 inch (2.54 cm) in loam top soil (Columbia, Mo.) that was sieved toremove large debris. Plants were grown at temperatures between 18-24° C.(65-75° F.) with 11 hours of light/day, and 50 ml of watering atplanting and every 3 days. After two weeks, plant heights and leafdiameters, as well as overall health of the plants were collected. Forgermination assays and determining 3 day root length, seeds were coatedas indicated above and evenly dispersed at 10 seeds per paper towel. Thepaper towels were wetted with 10 mls of water, rolled up, placed in asmall plastic bag and incubated at 30° C. or placed on a germinationheat mat at 27-30° C. (80-85° F.). Root measurements were recorded after3 days. Initial screening of rhizosphere isolates resulted in obtaininggreater than 100 distinct species of bacteria and fungi from therhizosphere. Some of the bacterial species are described in Table 16.Identified strains are indicated by their proper bacterialidentifications.

TABLE 16 Corn Seed Treatments Avg. Height Avg. Root Length (2 weeks) (3days) normalized to normalized to polymer control polymer controlBacterial Inoculant (%) (%) Polymer control 100 100   B. mycoides EE118111.1 189.1 B. subtilis EE148 99.4 172.8 Alcaligenes faecalis EE107111.5 129.2 B. mycoides EE141 109.2 143.5 B. mycoides BT46-3 105.6 141.3B. cereus family member EE128 105.6 — B. thuringiensis BT013A 101.8103.8 Paenibacillus massiliensis BT23 104.2 139.4 B. cereus familymember EE349 105.2 — B. subtilis EE218 106.6 — B. megaterium EE281 107.8—

Bacterial strains that produced the greatest effect on plant health aredescribed in Table 16. Bacterial strains were grown overnight in LuriaBertani broth at 37° C., and overnight cultures were spun down in acentrifuge. Media was decanted and the remaining bacterial pellet wassubjected to chromosomal DNA isolation using the Qiagen BacterialChromosomal DNA Isolation kit. Chromosomal DNA was subjected to PCRamplification of the 16S rRNA coding regions using the primers E338F5′-ACT CCT ACG GGA GGC AGC AGT-3′ (SEQ ID NO: 122), E1099R A 5′-GGG TTGCGC TCG TTG C-3′ (SEQ ID NO: 123), and E1099R B 5′-GGG TTG CGC TCG TTAC-3′ (SEQ ID NO: 124). PCR amplicons were purified using a Promega PCRpurification kit, and the resultant amplicons were diluted and sent tothe University of Missouri DNA Core for DNA sequencing. DNA sequenceswere compared to the NCBI BLAST database of bacterial isolates, andgenus and species were identified by direct comparison to known strains.Top identified species are indicated in Table 16. In many cases, 16SrRNA DNA sequences were only able to delineate the genus of the selectedbacterial strain. In cases where a direct identification was notforthcoming, additional biochemistry analyses, using methods standard inthe field, were performed to differentiate strains at the species andstrain levels, and the differentiated strains are listed in Table 17.

TABLE 17 B. B. cereus cereus B. family B. B. B. Paenibacillus B.Alcaligenes B. family B. thuringiensis member subtilis subtilismegaterium massiliensis mycoides faecalis mycoides member mycoides TestBT013A EE349 EE148 EE218 EE281 BT23 BT46-3 EE107 EE118 EE128 EE141Motility + + + + + + − + − − − Rhizoid Colony − − − − − + + − + − +Catalase + + + + + + + + + + + Oxidase + − − − − − − + − − − Nitrate + +wk − − − + + + + + Growth, 5% + wk − + + − + + − + − NaCl Growth, 7.5%wk − − + + − − − − − − NaCl Growth, 42° C. − + + + + + + + − + − Growth,50° C. − − − − − − − − − − − Growth, pH 5 wk − + + + − wk + − + −Growth, pH 9 + + − + + − wk + + + − Acid, − − wk + − + + wk + − wkCellobiose Acid, Lactose − + + + + − + + − + wk Acid, Starch − + − + +− + wk + + − wk = weak growth or low growth

Example 26. Testing of Plant-Growth Promoting Bacterial Strains onAlfalfa

The selected strains were grown in minimal media (KH₂PO₄ 3 g, Na₂HPO₄ 6g, NH₄Cl 1 g, NaCl 0.50 g, MgSO₄ 7H₂O 0.15 g, CaCl₂ 2H₂O 0.013 g, andglucose 1 g, per L dry weight). Overnight cultures (30° C.) of selectedstrains were spun down, media decanted off, and bacteria resuspended inan equal amount of distilled water. Ten Zeba-coated alfalfa seeds wereplanted for each treatment at a depth of 0.6 cm in loam top soil(Columbia, Mo.) that was sieved to remove large debris. Seeds wereinoculated at planting with 0.5 μl of resuspended bacteria in watermixed into 10 ml of H₂O. Ten ml of H₂O was sufficient to deliver thebacteria into the 3 in³ (7.62 cm³) of soil as well as saturate the soilfor proper germination of seeds. Plants were grown at temperaturesbetween 65-75° F. (18-24° C.) with 11 hours of light/day, and 5 ml ofwatering every 3 days. Alfalfa was allowed to grow for 1 week to analyzeemergence and initial outgrowth of plants under described conditions.Identified strains indicated by their proper bacterial identificationsand final height data are listed in Table 18.

TABLE 18 Alfalfa Avg. Height Bacterial Inoculant (cm) Comparison SEMUninoculated 4.82 — .008 B. aryabhattai CAP56 4.85 101.20% .016 B.nealsonii BOBA57 4.86 101.70% .021 E. cloacae CAP12 5.6 116.23% .020

Example 27. Testing of Plant-Growth Promoting Bacterial Strains onCucumbers

The selected strains were grown in minimal media (KH₂PO₄ 3 g, Na₂HPO₄ 6g, NH₄Cl 1 g, NaCl 0.50 g, MgSO₄ 7H₂O 0.15 g, CaCl₂ 2H₂O 0.013 g, andglucose 1 g, per L dry weight). Overnight cultures (30° C.) of selectedstrains were spun down, media decanted off, and resuspended in equalamount of distilled water. Ten cucumber seeds were planted for eachtreatment at a depth of 1 cm in loam top soil (Columbia, Mo.) that wassieved to remove large debris. Seeds were inoculated at planting with0.5 μl of resuspended bacteria in water mixed into 10 ml of H₂O. Ten mlof H₂O was sufficient to deliver the bacteria into the 3 in³ (7.62 cm³)of soil as well as saturate the soil for proper germination of seeds.Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11hours of light/day, and 5 ml of watering every 3 days. Cucumbers wereallowed to grow for 2 weeks to analyze emergence and initial outgrowthof plants under described conditions. Identified strains indicated bytheir proper bacterial identifications and final height data are listedin Table 19.

TABLE 19 Cucumbers Avg. Height Bacterial Inoculant (cm) Comparison SEMUninoculated 11.23 — .067 B. aryabhattai CAP53 11.5 102.00% .023 B.aryabhattai CAP56 11.35 101.20% .035 B. nealsonii BOBA57 11.33 101.10%.014

Example 28. Testing of Plant-Growth Promoting Bacterial Strains onYellow Squash

The selected strains were grown in minimal media (KH₂PO₄ 3 g, Na₂HPO₄ 6g, NH₄Cl 1 g, NaCl 0.50 g, MgSO₄ 7H₂O 0.15 g, CaCl₂) 2H₂O 0.013 g, andglucose 1 g, per L dry weight). Overnight cultures (30° C.) of selectedstrains were spun down, media decanted off, and resuspended in an equalamount of distilled water. Ten yellow squash seeds were planted for eachtreatment at a depth of 1 cm in loam top soil (Columbia, Mo.) that wassieved to remove large debris. Seeds were inoculated at planting with0.5 μl of resuspended bacteria in water mixed into 10 ml of H₂O. Ten mlof H₂O was sufficient to deliver the bacteria into the 3 in³ (7.62 cm³)of soil as well as saturate the soil for proper germination of seeds.Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11hours of light/day, and 5 ml of watering every 3 days. Squash wasallowed to grow for 2 weeks to analyze emergence and initial outgrowthof plants under described conditions. Identified strains indicated bytheir proper bacterial identifications, final height data, and finalleaf diameter (by span of the two leaves) data are listed in Table 20.

TABLE 20 Yellow Squash Avg. Leaf Height Diameter Bacterial Inoculant(cm) Comparison SEM (cm) Comparison Uninoculated 10.16 — .028 5.08 — B.aryabhattai CAP53 11.75 115.60% .055 7.25 142.60% B. flexus BT054 11.88116.90% .017 6.36 125.20% Bacillus mycoides 11.92 117.20% .051 6.33124.60% BT155 B. aryabhattai CAP56 11.95 117.60% .027 6.33 124.60% B.nealsonii BOBA57 11.89 117.00% .118 6.42 126.40% E. cloacae CAP12 11.42112.30% .039 6.83 134.40%

Example 29. Testing of Plant-Growth Promoting Bacterial Strains onRyegrass

The selected strains were grown in minimal media (KH₂PO₄ 3 g, Na₂HPO₄ 6g, NH₄Cl 1 g, NaCl 0.50 g, MgSO₄ 7H₂O 0.15 g, CaCl₂ 2H₂O 0.013 g, andglucose 1 g, per L dry weight). Overnight cultures (30° C.) of selectedstrains were spun down, media decanted off, and resuspended in an equalamount of distilled water. Thirty ryegrass seeds were planted for eachtreatment at a depth of 0.3 cm in loam top soil (Columbia, Mo.) that wassieved to remove large debris. Seeds were inoculated at planting with0.5 μl of resuspended bacteria in water mixed into 10 ml of H₂O. Ten mlof H₂O was sufficient to deliver the bacteria into the 3 in³ (7.62 cm³)of soil as well as saturate the soil for proper germination of seeds.Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11hours of light/day, and 5 ml of watering every 3 days. Ryegrass wasallowed to grow for 1.5 weeks to analyze emergence and initial outgrowthof plants under described conditions. Identified strains indicated bytheir proper bacterial identifications and height data are listed inTable 21.

TABLE 21 Ryegrass Avg. Height Bacterial Inoculant (cm) Comparison SEMUninoculated 1.61 — .023 B. aryabhattai CAP53 2.01 124.70% .012 B.flexus BT054 2.21 137.30% .034 Bacillus mycoides BT155 2.29 142.20% .049B. aryabhattai CAP56 2.19 136.00% .009 B. nealsonii BOBA57 2.29 142.40%.045 E. cloacae CAP12 1.98 122.50% .015

Example 30. Testing of Plant-Growth Promoting Bacterial Strains on Corn

The selected strains were grown in minimal media (KH₂PO₄ 3 g, Na₂HPO₄ 6g, NH₄Cl 1 g, NaCl 0.50 g, MgSO₄ 7H₂O 0.15 g, CaCl₂ 2H₂O 0.013 g, andglucose 1 g, per L dry weight). Overnight cultures (30° C.) of selectedstrains were spun down, media decanted off, and resuspended in an equalamount of distilled water. Ten corn seeds were planted for eachtreatment at a depth of 2.5 cm in loam top soil (Columbia, Mo.) that wassieved to remove large debris. Seeds were inoculated at planting with0.5 μl of resuspended bacteria in water mixed into 10 ml of H₂O. Ten mlof H₂O was sufficient to deliver the bacteria into the 3 in³ (7.62 cm³)of soil as well as saturate the soil for proper germination of seeds.Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11hours of light/day, and 5 ml of watering every 3 days. Corn was allowedto grow for 2 weeks to analyze emergence and initial outgrowth of plantsunder described conditions. Identified strains indicated by their properbacterial identifications and final height data are listed in Table 22.

TABLE 22 Corn Avg. Height Bacterial Inoculant (cm) Comparison SEMUninoculated 8.9 — .039 B. aryabhattai CAP53 11.01 123.60% .081 B.flexus BT054 9.96 112.00% .095 Bacillus mycoides strain BT155 9.6107.90% .041 B. aryabhattai CAP56 9.54 107.10% .088 B. nealsonii BOBA579.23 103.70% .077

Example 31. Testing of Plant-Growth Promoting Bacterial Strains onSoybeans

The selected strains were grown in minimal media (KH₂PO₄ 3 g, Na₂HPO₄ 6g, NH₄Cl 1 g, NaCl 0.50 g, MgSO₄ 7H₂O 0.15 g, CaCl₂ 2H₂O 0.013 g, andglucose 1 g, per L dry weight, or for Bradyrhizobium or Rhizobium onyeast mannitol media). Overnight cultures (30° C.) of selected strainswere spun down, media decanted off, and resuspended in equal amount ofdistilled water. Ten soybean seeds were planted for each treatment at adepth of 2.5 cm in loam top soil (Columbia, Mo.) that was sieved toremove large debris. Seeds were inoculated at planting with 0.5 μl ofresuspended bacteria in water mixed into 10 ml of H₂O. When testing twobacterial strains, 0.5 μl of each resuspended bacteria was mixed into 10ml of H₂O. Ten ml of H₂O was sufficient to deliver the bacteria into the3 in³ (7.62 cm³) of soil as well as saturate the soil for propergermination of seeds. Plants were grown at temperatures between 65-75°F. (18-24° C.) with 11 hours of light/day, and 5 ml of watering every 3days. Soybeans were allowed to grow for 2 weeks to analyze emergence andinitial outgrowth of plants under described conditions. Identifiedstrains indicated by their proper bacterial identifications and finalheight data are listed in Table 23. Co-inoculation of bacteria strainsin the present invention with members of the Bradyrhizobium sp. orRhizobium sp. lead to an increase in plant growth compared to eitherinoculant alone.

TABLE 23 Soybeans Avg. Height Bacterial Inoculant (cm) Comparison SEMUninoculated 13.94 — .089 B. aryabhattai CAP53 16.32 117.1% .146 B.flexus BT054 17.85 128.0% .177 Bacillus mycoides strain BT155 18.93135.8% .117 B. aryabhattai CAP56 17.23 123.6% .133 B. aryabhattai CAP5316.32 117.1% .077 B. aryabhattai CAP53 and 16.72 119.9% .182Bradyrhizobium sp. B. aryabhattai CAP53 and 17.32 124.2% .086 Rhizobiumsp. Bradyrhizobium sp. 14.25 102.2% Rhizobium sp. 14.75 105.8%

Example 32. Bacillus cereus Family Members with Plant Growth PromotingAttributes

Bacillus mycoides strain BT155, Bacillus mycoides strain EE118, Bacillusmycoides strain EE141, Bacillus mycoides strain BT46-3, Bacillus cereusfamily member strain EE349, Bacillus thuringiensis strain BT013A, andBacillus megaterium strain EE281 were grown in Luria Bertani broth at37° C. and overnight cultures were spun down, media decanted off, andresuspended in equal amount of distilled water. 20 corn seeds wereplanted for each treatment at a depth of 2.5 cm in loam top soil(Columbia, Mo.) that was sieved to remove large debris. Seeds wereinoculated at planting with 0.5 μl of resuspended bacteria in watermixed into 50 ml of H₂O. Fifty ml of H₂O was sufficient to deliver thebacteria into the 29 in³ (442.5 cm³) of soil as well as saturate thesoil for proper germination of seeds. Plants were grown at temperaturesbetween 65-72° F. with 13 hours of light/day, and 5 ml of watering every3 days. Seedlings were allowed to grow for 2 weeks to analyze emergenceand initial outgrowth of plants under described conditions. Identifiedstrains indicated by their proper bacterial identifications and finalheight data are listed in Table 24.

TABLE 24 Avg. Height, cm, Bacterial Inoculant Corn Percentage SEM, H2OControl 11.41   100% .123 B. mycoides EE118 12.43 108.9% .207 B.mycoides EE141 12.84 112.5% .231 B. mycoides BT46-3 11.81 103.5% .089Bacillus thuringiensis 12.05 105.6% .148 BT013A Bacillus cereus family13.12 114.9% .159 member EE128 Bacillus mycoides BT155 12.85 112.6% .163Bacillus megaterium EE281 11.99 105.1% .098All plant growth promoting bacteria tested had a beneficial effect oncorn height at two weeks under the described conditions. The Bacilluscereus family member EE128 strain had the greatest effect in this trial,giving a greater than at 14% boost in corn height.

Example 33. Enhanced Selection of Bacillus cereus Family Members toScreen for Plant Growth-Promoting and Other Beneficial Activities asBEMD Expression Host

The BEMD system can be used to display a wide range of proteins,peptides, and enzymes using any of the targeting sequences describedherein to provide beneficial agricultural effects. Additional beneficialeffects can be obtained by selecting an expression host (a Bacilluscereus family member) having inherent beneficial attributes. Manystrains of members of the Bacillus cereus family have plant-growthpromoting benefits. Additionally, many Bacillus cereus family memberstrains provide have protective effects, through direct fungicidal,insecticidal, nematocidal, or other protective activities. By using suchstrains these as the expression host for the BEMD system, the end sporeproduct would have a combination of positive benefits in agriculture.

Table 25 provides results for an experiments wherein a fusion proteinwas expressed in various Bacillus cereus family member strains. Allstrains are expressed a fusion protein comprising amino acids 1-35 ofSEQ ID NO: 1 and the phosphatase PhoA4 from Bacillus subtilis, abeneficial enzyme for enhanced phosphate uptake in corn. The gene wassynthesized, cloned into the pMK4 vector, and introduced into each ofthe Bacillus spp. indicated in Table 25 below. Strains were taken intosporulation by incubation at 30° C. on nutrient agar plates containingchloramphenicol 10 μg/ml for three days. Spores were collected, washed,and applied to corn at planting at a rate of 1×10⁵ CFU/ml in 50 ml ofwater per 7.62 cm diameter pot with 5 mg polyphosphate per pot. Corn wasgrown in silt loam soil for two weeks. Plants were grown under ideallight using T5 lamps, 54 watts, and exposed to 13 hours of light a dayunder controlled temperature conditions between 15.5-25.5° C. Plantswere watered to saturation every three days over a two week trial. Atthe end of two weeks, the height of each plant was measured andmeasurements were normalized to control Bacillus thuringiensis spores.Expression of the SEQ ID NO: 1—Phosphatase fusion protein led to anincrease in corn height at 2 weeks regardless of the expression hoststrain selected. As shown in Table 25, use of a plant-growth promotingBacillus cereus family member further increased corn height

TABLE 25 Height at 2 weeks, Bacillus Species Strain Fusion ProteinNormalized B. thuringiensis Strain BT013A None   100% B. thuringiensisStrain BT013A SEQ ID NO: 117.4% 1-Phosphatase B. mycoides Strain EE141None 107.3% B. mycoides Strain EE141 SEQ ID NO: 123.3% 1-Phosphatase B.cereus family Strain EE128 None 124.1% member B. cereus family StrainEE128 SEQ ID NO: 131.7% member 1-Phosphatase B. mycoides Strain BT155None 104.8% B. mycoides Strain BT155 SEQ ID NO: 121.9% 1-Phosphatase

Example 34. Use of Various Targeting Sequences to Expressβ-Galactosidase on the Surface of Bacillus thuringiensis

A wide variety of targeting sequences that that have a high degreehomology with amino acids 20-35 of BclA (amino acids 20-35 of SEQ IDNO: 1) can be used to display enzymes, proteins, and peptides on thesurface of Bacillus cereus family members. Several targeting sequenceswere compared by making fusion proteins containing the targetingsequences linked to Bacillus subtilis lipase. Fusion constructs weresynthesized using the promoters native to the targeting sequence, clonedinto the replicating plasmid pMK4, and introduced into Bacillusthuringiensis BT013A. Strains were taken into sporulation by incubationat 30° C. on nutrient agar plates containing chloramphenicol 10 μg/mlfor 3 days. Spores were collected, washed, and resuspended in PBS at arate of 1×10⁸/ml. 1×10⁵ spores for each fusion construct spores weresuspended in 400 μl dH₂O. The reactions were warmed with the reactioncomponents to the desired reaction temperature (40° C.). 200 μl workingbuffer was added (9:1 Solution A: Solution B). Solution A was 50 mM TrispH 10 and 13.6 mM deoxycholic acid and Solution B was 3 mg/mlp-nitrophenyl palmitate in isopropanol. The reaction was incubated at40° C. for 10 minutes and placed on ice, centrifuged to remove spores,and absorbance at 420 nm was recorded. The results are shown in Table 26below. Activity was normalized to a control fusion protein comprisingamino acids 1-35 of SEQ ID NO: 1 fused to Bacillus subtilis lipase.

TABLE 26 Strain Targeting sequence Enzyme Relative activity B.thuringiensis Amino acids 1-35 of Lipase   100% BT013A SEQ ID NO: 1 B.thuringiensis Amino acids 1-27 of Lipase  92.5% BT013A SEQ ID NO: 3 B.thuringiensis Amino acids 1-28 of Lipase  13.5% BT013A SEQ ID NO: 7 B.thuringiensis Amino acid 1-24 of Lipase  24.8% BT013A SEQ ID NO: 9 B.thuringiensis Amino acid 1-33 of Lipase  98.5% BT013A SEQ ID NO: 13 B.thuringiensis Amino acid 1-33 of Lipase 107.8% BT013A SEQ ID NO: 21 B.thuringiensis SEQ ID NO: 60 Lipase 137.1% BT013A B. thuringiensis SEQ IDNO: 62 Lipase 146.3% BT013A B. thuringiensis SEQ ID NO: 64 Lipase 115.7%BT013A B. thuringiensis SEQ ID NO: 68 Lipase  81.5% BT013A

Several targeting sequences linked to lipase result in higher expressionlevels and activity of enzyme on the surface of spores. In particular,SEQ ID NOs. 60, 62, and 64, each containing a shorter targetingsequence, resulted in enhanced fusion expression on the surface of theBEMD spores. All the fusion proteins containing targeting sequencestested resulted in surface display of lipase.

Example 35. Use of Various Exosporium Sequences to Express Lipase on theSurface of Bacillus thuringiensis and Demonstration of Fusion ProteinLocalization to the Exosporium Surface

A wide variety of exosporium proteins can be used to display enzymes,proteins, and peptides on the surface of Bacillus cereus family members.Several different exosporium proteins were compared by making fusionproteins containing the exosporium proteins linked to Bacillus subtilislipase as described in Example 34. Fusion constructs were synthesizedusing the promoter native to the exosporium protein indicated in Table27 below, cloned into the replicating plasmid pMK4, and introduced intoBacillus thuringiensis BT013A. Spores displaying the various exosporiumprotein-Bacillus subtilis 168 lipase fusions were made by growing thetransformed bacteria in brain heart infusion broth with selectivepressure from 10 μg/ml chloramphenicol, plating onto nutrient agarplates, and incubating at 30° C. for 3 days. After 3 days, the sporeswere washed off the plates, purified by centrifugation, and resuspendedin PBS at 1×10⁸ CFU/ml.

1×10⁵ spores for each fusion construct were resuspended in 400 μl dH₂O.The reactions were warmed with the reaction components to the desiredreaction temperature (40° C.). 200 μl of working buffer was added (9:1Solution A: Solution B). Solution A was 50 mM Tris pH 10 and 13.6 mMdeoxycholic acid and Solution B was 3 mg/ml p-nitrophenyl palmitate inisopropanol. The reaction was incubated at 40° C. for 10 minutes andplaced on ice, centrifuged to remove spores and absorbance at 420 nm wasrecorded. Results are shown in Table 27 below. Activity was normalizedto SEQ ID NO: 72 linked to lipase.

TABLE 27 Strain Exosporium protein Enzyme Relative activity B.thuringiensis SEQ ID NO: 72 Lipase  100% BT013A B. thuringiensis SEQ IDNO: 73 Lipase 134.5%  BT013A B. thuringiensis SEQ ID NO: 76 Lipase 17.8%BT013A B. thuringiensis SEQ ID NO: 80 Lipase 19.8% BT013A B.thuringiensis SEQ ID NO: 81 Lipase  8.2% BT013A

Use of the exosporium proteins of SEQ ID NOs. 72 and 73 resulted in thehighest enzyme activity on the spore. All the fusion proteins containingexosporium proteins resulted in surface display of active Bacillussubtilis 168 lipase, albeit at different levels.

Additional exosporium proteins were demonstrated to result in targetingof fusion proteins to the exosporium using the fluorescent reportermCherry. Fusion constructs were created that contained the exosporiumproteins of SEQ ID NOs. 74, 83, and 73 linked to the mCherry reporter.Spores were grown for 1.5 days, collected, and resuspended as describedabove. 7 μl of fluorescent spores were put under a Nikon E1000microscope and imaged during late sporulation. Circular localization ina ring is indicative of outer spore layer localization, and theappearance matches that of an exosporium protein. Fluorescent microscopyresults are shown in FIG. 2. FIGS. 2A, 2B, and 2C are fluorescentmicroscopy images of spores expressing fusion proteins comprising theexosporium proteins of SEQ ID NOs. 74, 83, and 73, respectively, and themCherry reporter. All three fusions demonstrated high levels offluorescence and exosporium localization, demonstrating their potentialutility for the expression of foreign proteins on the surface of theexosporium.

Example 36. Use of Various Targeting Sequences and Exosporium Proteinsto Express Phosphatase in Bacillus subtilis Spores and Effects of thePhosphatase-Expressing Spores in Soybeans

BEMD spores expressing Bacillus subtilis EE148 Phosphatase A4 (PhoA4)were created by gene synthesis of the genes coding for various targetingsequences and exosporium proteins under the control of their nativepromoters linked to PhoA4. The synthesized genes were cloned into pMK4and introduced into Bacillus thuringiensis BT013A. Spores displaying thevarious exosporium protein-Bacillus subtilis EE148 PhoA4 fusions weremade by growing the transformed bacteria in brain heart infusion brothwith selective pressure from 10 μg/ml chloramphenicol, plating ontonutrient agar plates, and incubating at 30° C. for three days. Afterthree days, the spores were washed off the plates, purified bycentrifugation, and resuspended in PBS at 1×10⁸ CFU/ml.

Soybeans were planted 2.54 cm deep in 10 cm deep pots filled withstandard loam topsoil. BEMD spores expressing PhoA4 were diluted to aconcentration of 1×10⁴/ml in 50 ml of water and applied to each plant atplanting. A water-only control was also included. Polyphosphate wasadded to pots in liquid at a rate of 0.5 mg/pot. Plants were grown underideal light using T5 lamps, 54 watts, and exposed to 13 hours of light aday under controlled temperature conditions between 15.5-25.5° C. Plantswere watered to saturation every three days over the two week trial. Atthe end of two weeks, the height of each plant was measured, andmeasurements were normalized to control water-only plants.

Results are shown in Table 28. Soy grown in the presence of BEMD sporesexpressing fusion proteins containing PhoA4 linked to various targetingsequences and exosporium proteins with different fusion partners withPhoA4 all exhibited enhanced growth, but the extent of the effect varieddepending on the targeting sequence or exosporium protein used.

TABLE 28 Targeting sequence or exosporium protein Height at 2 weeks,Bacillus species linked to PhoA4 Normalized H2O (No bacteria) N/A   100%Bacillus thuringiensis Amino acids 1-35 of   100% Strain BT013A SEQ IDNO: 1 Bacillus thuringiensis Amino acids 1-28 of 117.4% Strain BT013ASEQ ID NO: 3 Bacillus thuringiensis Amino acids 1-33 of 107.3% StrainBT013A SEQ ID NO: 21 Bacillus thuringiensis SEQ ID NO: 60 123.3% StrainBT013A Bacillus thuringiensis SEQ ID NO: 62 124.1% Strain BT013ABacillus thuringiensis SEQ ID NO: 72 131.7% Strain BT013A Bacillusthuringiensis SEQ ID NO: 73 104.8% Strain BT013A

Example 37. Co-Application of BEMD Spores and Seed Treatments, LiquidFertilizers, and Other Additives

BEMD spores expressing fusion proteins were tested for compatibilitywith various seed treatments. The BEMD spores expressed fusion proteinscomprising the targeting sequence of amino acids 1-35 SEQ ID NO: 1linked to a phosphatase (PhoA4) from Bacillus subtilis EE148 or thePOLARIS peptide. The synthesized genes were cloned into pMK4 andintroduced into Bacillus thuringiensis BT013A. Spores displaying thevarious exosporium protein-Bacillus subtilis EE148 PhoA4 or POLARISfusions were made by growing the transformed bacteria in brain heartinfusion broth with selective pressure from 10 μg/ml chloramphenicol,plating onto nutrient agar plates, and incubating at 30° C. for threedays. After three days, the spores were washed off the plates, purifiedby centrifugation, and resuspended in PBS at 1×10⁸ CFU/ml.

Plants were grown under ideal light using T5 lamps, 54 watts, andexposed to 13 hours of light a day under controlled temperatureconditions between 15.5-25.5° C. Plants were watered to saturation everythree days over the two week trial. At the end of two weeks, the heightof each plant was measured, and measurements were normalized to controlwater only plants. Results are shown in Table 29 below. Drench=appliedto soil at 50 ml per pot. Polymer=ACCELERON seed coating polymer only.BEMD spores were added at 1×10⁴ cells/50 ml for drench applications.BEMD spores were added at 1.3×10⁴/cells/seed for seed coatingapplications. 10-34-0 and 6-24-6 are standard commercial starterfertilizer compositions. 10-34-0 is liquid ammonium phosphate. 6-24-6 islow salt liquid phosphate fertilizer with an ortho/poly formulation.Colorant=Becker Underwood red seed coating coloring agent. MACHO, APRON,and CRUISER are commercial fungicides used on seeds. MACHO contains theactive ingredient imidacloprid, APRON contains the active ingredientmefenoxam, and CRUISER contains a mixture of the active ingredientsthiamethoxam, mefenoxam, and fludioxonil. The spores were found to becompatible with many seed applications and retained their ability tostimulate plant growth in corn.

TABLE 29 Corn height at 2 BEMD treatment Chemical weeks, normalized NoneNone (Water Drench)   100% None Polymer Only 101.3% BEMD PhoA4 N/A(Drench) 111.3% BEMD POLARIS N/A (Drench) 106.7% BEMD PhoA4 Polymer109.3% BEMD POLARIS Polymer 107.3% BEMD PhoA4 Polymer + Colorant 102.3%BEMD PhoA4 Polymer + MACHO 107.9% BEMD PhoA4 Polymer + APRON 112.3% BEMDPhoA4 Polymer + CRUISER 116.8% BEMD PhoA4 Polymer + Colorant + 113.7%MACHO + APRON + CRUISER None 10-34-0 Starter 108.5% (Drench) BEMD PhoA410-34-0 Starter 114.7% Fertilizer (Drench) None 6-24-6 Starter 102.6%Fertilizer (Drench) BEMD PhoA4 6-24-6 Starter 112.9% Fertilizer (Drench)

BEMD spores were found to be compatible with all seed coating amendmentstested. There was a slight decrease in activity when BEMD PhA4 sporeswere combined with colorant and polymer alone, but the spores regainedfull activity with colorant in combination with other fungicides. BEMDspores also worked well with liquid fertilizers. Starter fertilizerscontributed to plant growth most likely through direct nutrientsupplementation. BEMD spores worked with both starter fertilizers,suggesting that phosphatase activity can still lead to increased plantgrowth in the presence of excess nutrients. Combinations of BEMD sporeswith fungicides exhibited greater increases in plant growth than BEMDspores alone, likely due to protection given to young corn plants duringearly growth.

Example 38. The Use of the BEMD Spores as a Foliar Addition for ReducingStress Inhibition of Growth on Corn

The BEMD spore display system can be used to deliver enzymes that canalleviate some stress from growing plants in the field or greenhouse. Toaccomplish this, enzymes were selected that selectively act uponreactive oxygen species in soil. Reactive oxygen species are a keymarker of stress in plants.

BEMD spores expressing fusion proteins comprising the targeting sequenceof amino acids 1-35 of SEQ ID NO: 1 linked to chitosinase, superoxidedismutase, catalase, or β1,3 glucanase from Bacillus thuringiensisBT013A were generated. The synthesized genes were cloned into pMK4 andintroduced into Bacillus thuringiensis BT013A. Spores displaying thevarious protein fusions were made by growing the transformed bacteria inbrain heart infusion broth with selective pressure from 10 μg/mlchloramphenicol, plating onto nutrient agar plates, and incubating at30° C. for three days. After three days, the spores were washed off theplates, purified by centrifugation, and resuspended in PBS at 1×10⁸CFU/ml.

Three week old corn plants at the V5 stage were grown under ideal lightusing T5 lamps, 54 watts, and exposed to 13 hours of light a day undercontrolled temperature conditions between 15.5-25.5° C. Plants werewatered to saturation every three days over the course of the trial. Asthe plants reach V5, BEMD spores or positive control chemicals weresprayed on the leaves at either 1×10⁵ BEMD spores/ml or at therecommended rates for the chemicals. A total of 1 ml of spray wasapplied to each plant individually. Plant heights were taken just priorto the application of the foliar sprays. The corn plants were thenstressed by warming to 32.2° C. and decreasing watering to once perweek. Plants were kept under stressed conditions for two weeks. At theend of the two weeks, plant heights were again measured, and visualappearance recorded. Under these stressed conditions, plant growth wasminimal in control treatments. The ability to continue to grow understressed conditions was measured by an increase in plant height over thetwo week span as compared to the water-only control. Results are shownin Table 30 below.

TABLE 30 Change in plant Height over 2 week Treatment Rate stress NoneNone   0% Bacillus thuringiensis 1 ml/plant −1.6%   BT013A spores BEMDChitosinase 1 ml/plant 0.3% BEMD Chitosinase 1 ml/plant and 4.7% andChitosan 5 mM BEMD Superoxide 1 ml/plant 8.3% Dismutase BEMD B1,3 1ml/plant 4.9% Glucanase Salicylic Acid 1 ml/plant 5.8% Benzothiadiazole1 ml/plant 7.3% (BTH) BEMD Catalase 1 ml/plant −0.5%  

Several destressing enzymes were applied to corn using the BEMD system,as shown in Table 30 above. Control spores had no significant effect(decrease in plant height of −1.6%. The BEMD chitosinase enzyme had apositive effect when combined with its substrate, chitosan. The two bestperforming enzymes were BEMD β-1,3-glucanase and BEMD superoxidedismutase. BEMD β-1,3-glucanase has a primarily antifungal activity, butcan also have direct effects on plants. Salicylic acid and BTH werepositive controls for the foliar assay, and positive responses were seenfor both. This foliar delivery method can be used for deliveringdestressing enzymes to the plants at various times of the season.

Example 39. Expression Levels of Fusion Proteins Using Various Sigma-KContaining Promoters

As shown in Example 23 above, replacing native promoter of a targetingsequence, exosporium protein, or exosporium protein fragment can greatlyaffect the level of fusion protein expressed on the exosporium of aBacillus cereus family spore. For example, replacing the native BclApromoter with the BclB promoter greatly reduces the level of fusionprotein on the surface of Bacillus cereus family member spores.Alternatively, replacement of native BclB promoter with the BclApromoter increases fusion protein levels on the exosporium dramatically.

Relative promoter expression levels for various exosporium proteinsunder the control of their native sporulation promoters were obtainedfrom microarray data from Bergman et al., 2008. The relative expressionlevels were determined during late sporulation timing (300 minutes afterthe start of the experiment), when sigma K promoters are most active.Sigma K promoters are key promoters for expression of exosporiumlocalized genes and associated proteins. Relative expression is theincrease in a gene's expression level when compared to the average ofall other genes of the chromosome at all given times. Table 31 belowshows the relative expression levels of a variety of sigma K drivengenes in Bacillus cereus family members.

TABLE 31 Relative Expression Protein (Promoter SEQ ID NO.) (Foldincrease in mRNA) CotY (SEQ ID NO: 97) 79.21 Rhamnose Promoters (SEQ IDNO: 96) 75.69 BclC (SEQ ID NO: 98) 14.44 Sigma K (SEQ ID NO: 99) 64 BclAadjacent US Glycosyl transferase 72.25 promoter 1 (SEQ ID NO: 101) BclAadjacent DS Glycosyl transferase 73.96 promoter 2 (SEQ ID NO: 102) BclA(SEQ ID NO: 85) 77.44 ExsY (SEQ ID NO: 91) 32.49 YjcA (SEQ ID NO: 93) 64YjcB (SEQ ID NO: 94) 70.56 BxpB/ExsFA (SEQ ID NO: 95) 30.25 InhA (SEQ IDNO: 100) 34.25

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above fusion proteins, Bacilluscereus family members, formulations, and methods without departing fromthe scope of the invention, it is intended that all matter contained inthe above description and shown in the accompanying drawing shall beinterpreted as illustrative and not in a limiting sense.

What is claimed is:
 1. A recombinant Bacillus cereus family member thatexpresses a fusion protein, wherein the fusion protein comprises: atargeting sequence, exosporium protein, or exosporium protein fragmentselected from the group consisting of: (a) an amino acid sequence havingat least 43% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least 54%; (b) a targetingsequence comprising amino acids 1-35 of SEQ ID NO: 1; (c) a targetingsequence comprising amino acids 20-35 of SEQ ID NO: 1; (d) a targetingsequence comprising SEQ ID NO: 1; (e) a targeting sequence comprisingSEQ ID NO: 60; (f) a targeting sequence comprising amino acids 22-31 ofSEQ ID NO: 1; (g) a targeting sequence comprising amino acids 22-33 ofSEQ ID NO: 1; and (h) a targeting sequence comprising amino acids 20-31of SEQ ID NO: 1; and an aminocyclopropane-1-carboxylic acid deaminase.2. The recombinant Bacillus cereus family member of claim 1, wherein thetargeting sequence comprises an amino acid sequence having at least 50%identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identitywith amino acids 25-35 is at least 63%.
 3. The recombinant Bacilluscereus family member of claim 1, wherein the targeting sequencecomprises an amino sequence having at least 62% identity with aminoacids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35is at least 72%.
 4. The recombinant Bacillus cereus family member ofclaim 1, wherein the targeting sequence comprises an amino acid sequencehaving at least 81% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 90%. 5.The recombinant Bacillus cereus family member of claim 1, wherein thetargeting sequence consists of: (a) an amino acid sequence consisting of16 amino acids and having at least about 43% identity with amino acids20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is atleast 54%; (b) amino acids 1-35 of SEQ ID NO: 1; (c) amino acids 20-35of SEQ ID NO: 1; (d) SEQ ID NO: 1; or (e) SEQ ID NO:
 60. 6. Therecombinant Bacillus cereus family member of claim 1, wherein thetargeting sequence comprises SEQ ID NO:
 60. 7. The recombinant Bacilluscereus family member of claim 1, wherein the targeting sequence,exosporium protein, or exosporium protein fragment further comprises amethionine residue at the amino acid position immediately preceding thefirst amino acid of the targeting sequence, exosporium protein, orexosporium protein fragment.
 8. The recombinant Bacillus cereus familymember of claim 1, wherein targeting sequence, the exosporium protein,or the exosporium protein fragment comprises the amino acid sequence GXTat its carboxy terminus, wherein X is any amino acid.
 9. The recombinantBacillus cereus family member of claim 1, wherein the fusion proteinfurther comprises an amino acid linker between the targeting sequence,the exosporium protein, or the exosporium protein fragment and theaminocyclopropane-1-carboxylic acid deaminase.
 10. The recombinantBacillus cereus family member of claim 9, wherein the linker comprises apolyalanine linker, a polyglycine linker, or a linker comprising amixture of both alanine and glycine residues.
 11. The recombinantBacillus cereus family member of claim 1, wherein the recombinantBacillus cereus family member comprises Bacillus anthracis, Bacilluscereus, Bacillus thuringiensis, Bacillus mycoides, Bacilluspseudomycoides, Bacillus samanii, Bacillus gaemokensis, Bacillusweihenstephensis, or a combination thereof.
 12. The recombinant Bacilluscereus family member of claim 1, wherein the recombinant Bacillus cereusfamily member comprises a plant-growth promoting strain of bacteria. 13.The recombinant Bacillus cereus family member of claim 1, wherein therecombinant Bacillus cereus family member is inactivated.
 14. Aformulation comprising a recombinant Bacillus cereus family member ofclaim 1 and an agriculturally acceptable carrier.
 15. The formulation ofclaim 14, wherein the formulation further comprises at least oneagrochemical.
 16. The formulation of claim 15, wherein the agrochemicalcomprises a fertilizer, a micronutrient fertilizer material, aninsecticide, a herbicide, a fungicide, a molluscicide, an algicide, aplant growth amendment, a bacterial inoculant, a fungal inoculant, or acombination thereof.
 17. The formulation of claim 16, wherein theagrochemical comprises the fertilizer.
 18. The formulation of claim 17,wherein the fertilizer comprises ammonium sulfate, ammonium nitrate,ammonium sulfate nitrate, ammonium chloride, ammonium bisulfate,ammonium polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrousammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate,calcium ammonium nitrate, calcium sulfate, calcined magnesite, calciticlimestone, calcium oxide, calcium nitrate, dolomitic limestone, hydratedlime, calcium carbonate, diammonium phosphate, monoammonium phosphate,magnesium nitrate, magnesium sulfate, potassium nitrate, potassiumchloride, potassium magnesium sulfate, potassium sulfate, sodiumnitrate, magnesian limestone, magnesia, urea, urea-formaldehyde, ureaammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylidenediurea, K₂SO₄-2MgSO₄, kainite, sylvinite, kieserite, Epsom salts,elemental sulfur, marl, ground oyster shells, fish meal, oil cakes, fishmanure, blood meal, rock phosphate, superphosphate, slag, bone meal,wood ash, manure, bat guano, peat moss, compost, green sand, cottonseedmeal, feather meal, crab meal, fish emulsion, humic acid, or acombination thereof.
 19. A plant seed treated with a recombinantBacillus cereus family member of claim
 1. 20. The plant seed of claim19, wherein the plant seed is coated with the recombinant Bacilluscereus family member.
 21. A plant seed treated with a formulation ofclaim
 14. 22. The plant seed of claim 21, wherein the plant seed iscoated with the formulation.